Acid-Degradable and Bioerodible Modified Polyhydroxylated Materials

ABSTRACT

Compositions and methods of making a modified polyhydroxylated polymer comprising a polyhydroxylated polymer having reversibly modified hydroxyl groups, whereby the hydroxyl groups are modified by an acid-catalyzed reaction between a polydroxylated polymer and a reagent such as acetals, aldehydes, vinyl ethers and ketones such that the modified polyhydroxylated polymers become insoluble in water but freely soluble in common organic solvents allowing for the facile preparation of acid-sensitive materials. Materials made from these polymers can be made to degrade in a pH-dependent manner. Both hydrophobic and hydrophilic cargoes were successfully loaded into particles made from the present polymers using single and double emulsion techniques, respectively. Due to its ease of preparation, processability, pH-sensitivity, and biocompatibility, of the present modified polyhydroxylated polymers should find use in numerous drug delivery applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/079,091, filed on Jul. 8, 2008, and to International ApplicationNo. PCT/US2009/049415, filed on Jul. 1, 2009, both of which are herebyincorporated by reference in their entirety.

This application is related to and incorporates by reference U.S.Provisional Patent Application No. 60/798,177, filed on May 5, 2006.This application is also related to and incorporates by referenceco-pending divisional U.S. patent application Ser. No. 11/388,924, filedon Mar. 28, 2006.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made during work partially supported by NationalInstitutes of Health under Grant RO1GM44885-16 and Grant RO1 EB005824and the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.The government has certain rights in this invention.

REFERENCE TO SEQUENCE LISTING

This application also incorporates by reference the attached sequencelisting containing cellular targeting sequences in electronic and paperform, hereby certified as identical copies.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention generally relates to the field of acid-degradable andbioerodible materials and polymers for use in delivery of bioactivematerials such as antigens, DNA and other therapeutics or as bulkmaterials such as sutures, scaffolds, and implants.

2. Description of the Related Art

Polyesters, polyorthoesters, and polyanhydrides are widely usedmaterials for biomedical applications due to their biodegradability,biocompatibility and processability (Yolles, S.; Leafe, T. D.; Meyer, F.J., J. Pharm. Sci. 1975, 64, 115-6; Heller, J., Ann. N. Y Acad. Sci.1985, 446, 51-66; Rosen, H. B.; Chang, J.; Wnek, G. E.; Linhardt, R. J.;Langer, R., Biomaterials 1983, 4, 131-3). Microparticles made from thesepolymers have been used as carriers for vaccine applications, genedelivery and chemotherapeutic agents.(Solbrig, C. M.; Saucier-Sawyer, J.K.; Cody, V.; Saltzman, W. M.; Hanlon, D. J., Mol. Pharm. 2007, 4,47-57; Gvili, K.; Benny, O.; Danino, D.; Machluf, M., Biopolymers 2007,85, 379-91; Sengupta, S.; Eavarone, D.; Capila, I.; Zhao, G. L.; Watson,N.; Kiziltepe, T.; Sasisekharan, R., Nature 2005, 436, 568-572). Theencapsulated cargo is typically released over the course of severalmonths via surface erosion and the slow degradation of thepolymer.(Matsumoto, A.; Matsukawa, Y.; Suzuki, T.; Yoshino, H., J.Control Release 2005, 106, 172-80).

For many drug delivery applications, it is desirable to releasetherapeutic agents under mildly acidic conditions, which can be foundfor example in sites of inflammation, lysosomal compartments, or tumortissue. ((a) Sun-Wada, G. H.; Wada, Y.; Futai, M., Cell Struct. Funct.2003, 28, 455-63 (b) Helmlinger, G.; Sckell, A.; Dellian, M.; Forbes, N.S.; Jain, R. K., Clin. Cancer Res. 2002, 8, 1284-91) Acid-sensitiveliposomes, micelles and hydrogels ((a) Sawant, R. M.; Hurley, J. P.;Salmaso, S.; Kale, A.; Tolcheva, E.; Levchenko, T. S.; Torchilin, V. P.,Bioconjug Chem 2006, 17, 943-9 (b) Mandracchia, D.; Pitarresi, G.;Palumbo, F. S.; Carlisi, B.; Giammona, G., Biomacromolecules 2004, 5,1973-82 (c) Murthy, N.; Thng, Y. X.; Schuck, S.; Xu, M. C.; Frechet, J.M. J., J. Am. Chem. Soc. 2002, 124, 12398-12399) have previously beendeveloped, but few easily-prepared polymeric materials exist thatcombine acid-sensitivity and biodegradability.

Poly(β-amino esters), which are protonated and thus become soluble atlower pH (Little, S. R.; Lynn, D. M.; Ge, Q.; Anderson, D. G.; Puram, S.V.; Chen, J.; Eisen, H. N.; Langer, R., Proc. Natl. Acad. Sci. U. S. A.2004, 101, 9534-9), constitute one such material. However, thesepolymers become polycationic under acidic conditions and must be blendedwith biocompatible polyesters to reduce their toxicity (Little, S. R.;Lynn, D. M.; Puram, S. V.; Langer, R., J. Control Release 2005, 107,449-62).

Currently there is no system with the flexibility and biocompatibilityof polyester materials, but with the additional benefit of a change inrate of payload release that is sensitive to physiologically relevantacidic conditions.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to bioerodible modifiedpolyhydroxylated polymers for application in the delivery of proteins,vaccines, drugs (such as the anticancer drugs cisplatin, paclitaxel ortaxotere), and other bioactive materials. In one embodiment, themodified polyhydroxylated polymer comprising a polyhydroxylated polymerwith reversibly modified hydroxyl groups, wherein the hydroxyl groupsare modified by a one-step reaction to feature a functional groupselected from the group consisting of acetals, aromatic acetals, andketals.

In one preferred embodiment, the hydroxyl groups in the polyhydroxylatedpolymers are modified, thereby rendering the modified polyhydroxylatedpolymer acid- degradable, pH sensitive and insoluble in water.

In a preferred embodiment, the modified polyhydroxylated polymers arealso acid-degradable comprising acid-degradable modifiedpolyhydroxylated polymers that are designed to deliver bioactivematerials. In one embodiment, the modified polyhydroxylated polymersdeliver bioactive materials upon hydrolysis of an acetal or ketallinkage at pH 5 to pH 7.4. In one embodiment, the polymer compositionsare made using polyhydroxylated polymers resulting in modifiedpolyhydroxylated polymers containing an acid-degradable linkage, whichhydrolyzes to release and deliver bioactive material. In anotherembodiment, the modified polyhydroxylated polymers are bioerodiblewhereby degradation of the polymers allows for slow release of anybioactive material to be delivered.

The polymers may be processed to form particles, bulk materials orimplants for the pH dependent controlled release of small drug orbiotherapeutics. These polymers could also be used as vehicles for drugconjugation or complexation designed to release their drug at mild pHvalues or scaffolds for tissue engineering purposes.

The polymers of the current invention are designed to degrade intonatural polyhydroxylated products, releasing their contents in responseto the mildly acidic conditions found in lysosomes, tumors, andinflammatory tissues. In one embodiment, the present polymers willhydrolyze at a preferred pH range of 4.5 to 6.8, more preferably pH 5.0to 6.0. Preferably, the polymers will completely hydrolyze within 24hours at pH 5.0, or conditions such as in the lysosome, and releasetheir encapsulated or bound contents after entering a cell.

In one embodiment, the polyhydroxylated polymers are preformed naturalpolymers or hydroxyl-containing polymers including but not limited to,multiply-hydroxylated polymers, polysaccharides, carbohydrates, polyols,polyvinyl alcohol, poly amino acids such as polyserine, and otherpolymers such as 2-(hydroxyethyl)methacrylate.

In one embodiment, the polysaccharides that can be used include but arenot limited to, dextran, mannan, pullulan, maltodextrin, starches,cellulose and cellulose derivatives, gums (e.g., xanthan, locust bean,etc.), and pectin. In one embodiment, the polysaccharides are dextran ormannan.

In another embodiment, the modified polysaccharides have pendantacetals, thus providing acetal-derivatized polysaccharides. In oneembodiment, the modified polyhydroxylated polymers areacetal-derivatized dextran, acetal-derivatized mannan oracetal-derivatized polyvinyl alcohols.

In one embodiment, the reversible modification of the polyhydroxylatedpolymer to produce the present acid-degradable and bioerodible modifiedpolyhydroxylated polymers is performed in a one-step modificationprocess. The one-step reversible modification of the hydroxyl groups canbe carried out to provide modified hydroxyl groups, wherein at least20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or100% of the hydroxyl groups in the polymer are modified.

In one embodiment, polyhydroxylated polymers are prepared and reactedwith a functionalizing group and result in a variety of pH sensitive andfunctionalized polyhydroxylated polymers with different solubilities.

This class of polymers are simple to prepare and completely degradable.Select polymers were characterized. The degradation of these polymersinto small molecules was monitored at pH 7.4 and pH 5 over time alongwith methods of controlling the rate of degradation, thus making thesepolymers promising candidates for drug delivery systems.

A method of preparing a modified polyhydroxylated acid-degradablecomposition for delivering a bioactive material to a cell, comprisingthe steps of (a) preparing a mixture which contains a polyhydroxylatedpolymer and a functional group, wherein a one-step reaction provides amodified polyhydroxylatedpolymer having modified hydroxyl groupscontaining an acid-degradable linkage; (b) forming particles of thepolymer in the presence of a bioactive material; and (c) recovering theresulting polymer particles having bioactive material bound or entrappedthereto.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows previous acid-degradable systems (top and middle) comparedto the present system (bottom).

FIG. 2 shows a general synthetic scheme for the preparation of modifiedpolyhydroxylated polymers.

FIG. 3 shows an overall synthetic scheme for the preparation of modifiedpolydroxylated polymers.

FIG. 4 shows the scheme for synthesis of dextran modified to dextranhaving cyclic and acyclic ketals masking the hydroxyl groups.

FIG. 5 shows water-soluble dextran modified to organic-soluble dextranhaving cyclic and acyclic acetals masking the hydroxyl groups and anSEMimage of the Ac-DEX particles.

FIG. 6 shows the synthesis of acetal-modified dextran (Ac-DEX) andparticle formation (i) 2-methoxypropene, pyridinium-p-toluenesulfonate,DMSO (ii) solvent-evaporation-based particle formation (scale bar is 2μm).

FIG. 7 shows the scheme for synthesis of dextran modified to dextranhaving aliphatic acetals masking the hydroxyl groups.

FIG. 8 shows the scheme for synthesis of dextran modified to dextranhaving aromatic acetals masking the hydroxyl groups.

FIG. 9 shows the scheme for synthesis of pre-functionalized dextranhaving alkyne functional groups or a dye to prefunctionalized dextranhaving cyclic and acyclic ketals masking the hydroxyl groups.

FIG. 10 shows the scheme for the synthesis of mannan modified to mannanhaving cyclic and acyclic ketals masking the hydroxyl groups and an SEMof particles formed by a solvent-evaporation-based technique (scale baris 1 μm).

FIG. 11 shows the synthetic scheme of acetalated polyvinyl alcohol using2-methoxypropene, pyridinium-p-toluenesulfonate, DMSO.

FIG. 12. Representative SEM image of (A) Ac-DEX particles, (C)singleemulsion Ac-DEX particles and (D) single emulsion acetalated mannanparticles. FIG. 12B shows time-lapse photos of Ac-DEX particles underphysiological or acidic conditions

FIG. 13 dissolution halflife at pH 5 vs dextran reaction time.

FIG. 14 (a) Dissolution of dextran from Ac-DEX particles in either pH 5or pH 7.4 buffer at 37° C. (b) Normalized ¹H-NMR data from thedegradation of Ac-DEX particles at pH 5.5 and 37° C. showingintegrations of signals corresponding to acetone, methanol and acetalgroups. (c) Time-lapse photos of Ac-DEX particles under physiological oracidic conditions.

FIG. 15 shows a graph of the results of the B3Z assay measuring antigenpresentation of RAW macrophages pulsed with free OVA or Ac-DEX particlesencapsulating OVA.

FIG. 16. Size distribution histograms of (a) double emulsion particlesencapsulating OVA or (b) single emulsion particles encapsulating pyrene.The results in the text are presented as average particle diameters±halfwidth of the distribution at half maximal height

FIG. 17. Release profile of FITC-dextran encapsulated in Ac-DEXparticles at 37° C. and in pH 5 or pH 7.4 buffer.

FIG. 18. Stack plot of ¹H NMR spectra of empty Ac-DEX particlesincubated in deuterated pH 5.5 buffer over time. Spectra are shown forthe first eight days and are normalized with respect to the integrationof the TMS peak.

FIG. 19. Final ¹H NMR spectrum of degraded Ac-DEX particles.

FIG. 20. Cell viability of RAW macrophages was measured by MTT assayafter overnight culture with (a) Ac-DEX particles or PLGA particles or(b) Ac-DEX particle degradation products.

FIG. 21. Particles made from Ac-DEXwere (a) modified at their reducingends through oxime linkages using cell penetrating peptides (CPP)containing an aminoxy group . HeLa cells (b) were incubated withfluorescently labeled Ac-DEX particles that were either (c) unmodifiedor (d) modified with CPP groups. Modification with CPPs led tosignificantly enhanced uptake of particles relative to unmodifiedparticles.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Definitions

The term “bioactive material” herein refers to a composition having aphysiological effect on a cell, such as a protein, antigen, polypeptide,polynucleotide, an enzyme or other organic molecule, for example, drugsor chemotherapeutics.

The terms “nucleotide”, “oligonucleotide”, and “polynucleotide” hereinrefer to single- or multi-stranded deoxyribonucleotides (DNA), single-or multi-stranded ribonucleotides (RNA), or single-or multi-strandedpeptide nucleic acids (PNA).

The term “acetal” herein refers to a geminal diether in which both etheroxygens are bound to the same carbon.

The term “aryl” herein refers to a homocyclic aromatic, whether or notfused, having 6 to 12 carbon atoms optionally substituted with one tothree substituents, wherein said substituents are preferably N or O, orunsubstituted.

The term “alkyl” herein refers to an aliphatic linear or branched chainunivalent groups of the general formula C_(n)H_(2n+1) derived fromaliphatic hydrocarbons such as methyl CH₃, ethyl C₂H₅, propyl C₃H₇,2-methyl propyl C₄H₁₁, and the like or cyclic aliphatic univalent groupsof the general formula C_(n)H_(2n−1) derived from cyclic aliphatichydrocarbons, such as cyclypropyl C₃H₅, cyclopentyl C₅H₉ and the like ,where n is between 2 and 20.

The term “loading” herein refers to the amount of bioactive materialthat is encapsulated per milligram of the drug delivery systems. Thismay be expressed in terms of μg material/mg drug delivery system, onaverage, based on the starting bioactive material/polymers ratio.

The term “loading efficiency” herein refers to the percentage of thestarting amount of bioactive material that is actually encapsulated.

The term “ketal” herein refers to an acetal in which the central carbonbound to two oxygen atoms is bound to two alkyl groups.

The terms “d”, “min”, “s” and “rt” used herein refer to days, minutes,seconds, and room temperature, respectively.

Introduction

In one embodiment, the present invention provides a modifiedpolyhydroxylated polymer comprising a polyhydroxylated polymer havingreversibly modified hydroxyl groups, wherein the hydroxyl groups aremodified by a one-step reaction to feature a functional group selectedfrom the group consisting of acetals, aromatic acetals, ketals.

In one preferred embodiment, the hydroxyl groups in the polyhydroxylatedpolymers are modified, thereby rendering the modified polyhydroxylatedpolymer acid degradable, pH sensitive and insoluble in water.

Thus, the present invention describes a system with the flexibility andbiocompatibility of polyester materials, but with the additional benefitof a change in rate of hydrolysis or degredation that is sensitive tophysiologically relevant acidic conditions. Thus in one embodiment, asolubility switching mechanism is used in which a biocompatible,water-soluble (polyhydroxylated) polymer may be reversibly modified tomake it insoluble in water, but soluble in organic solvents. Materialsmade from the modified polyhydroxylated polymer could then be degradedunder the specific conditions that reverse the original modification.

In one embodiment, hydroxyl groups displayed on the polyhydroxylatedpolymer backbone are modified to display a functional group having anacetal or ketal linkage therein. This group is designed to remainlargely stable in plasma at neutral physiological pH (about 7.4), butdegrade intracellularly by hydrolysis in the more acidic environment ofthe endosome or lysosome (about pH 5.0-6.0). The modifiedpolyhydroxylated polymers exhibit hydrolysis and degradation, wherebythe resulting degradation products are the polyhydroxylated polymer andthe small molecule byproducts.

In a preferred embodiment, the modified polyhydroxylated polymers areprocessed to deliver a bioactive material. In a preferred embodiment,polymer particles hydrolyze under acidic conditions and release thebioactive material in response to the mildly acidic conditions, found inthe body such as in tumors, inflammatory tissues and in cellularcompartments such as lysosomes and phagolysosomes of antigen presentingcells.

In a preferred embodiment, the bioactive material includes but is notlimited to, antigens, proteins, polynucleotides, polypeptides, peptoids,small drug molecules and other bioactive material.

A. Polyhydroxylated Polymers

In one embodiment, the polyhydroxylated polymers are preformed naturalpolymers or hydroxyl-containing polymers including but not limited to,multiply-hydroxylated polymers, polysaccharides, carbohydrates, polyols,polyvinyl alcohol, poly amino acids such as polyserine, and otherpolymers such as 2-(hydroxyethyl)methacrylate.

In one embodiment, the polysaccharides that can be used include but arenot limited to, dextran, mannan, pullulan, maltodextrin, starches,cellulose and cellulose derivatives, gums (e.g., xanthan, locust bean,etc.), and pectin. In one embodiment, the polysaccharides are dextran ormannan.

In one embodiment, the modified polyhydroxylated polymers are preparedby a single one-step reaction. The hydroxyl groups in thepolyhydroxylated polymer are modified to feature a functional groupselected from the group consisting of acetals, aromatic acetals, ketals,vinyl ethers, aldehydes and ketones. Typically the modification processinvolves an acid-catalyzed reaction between a polyhydroxylated polymerand functional moleculessuch as vinyl ethers, acetals, aldehydes, orketones

In one embodiment, the reversible modification of the hydroxyl groupsshould be carried out to provide modified hydroxyl groups, wherein atleast 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%,99% or 100% of the hydroxyl groups in the polymer are modified. In oneembodiment, at least 20-85% of the hydroxyl groups are modified. Inanother embodiment, at least 75-85% of the hydroxyl groups are modified.

In general, the choice of the polyhydroxylated polymer and the degree ofmodification also reflects such factors as ease of synthesis,solubility, commercially available reagents, the type of acid-degradablepolymer desired, the loading efficiency, dispersion of drug deliverysystems comprised of the polymers, toxicity and the hydrolysis rates ofthe acetal linkage.

In a preferred embodiment, the degradation products are biocompatibleand biodegradable. In another embodiment, the degradation products aresmall molecules as well as polymers with a molecular mass of up to10,000 daltons or lower, more preferably 1000 daltons, and mostpreferably 400 daltons or lower. In a preferred embodiment, thedegradation product(s) should be non-immunogenic and non-toxic, forexample, with the size and/or toxicity levels preferred by one havingskill in the art for approved in vivo use.

In another embodiment, the modified polyhydroxylated polymers aremodified polysaccharides with pendant acetals, thus providingacetal-derivatized polysaccharides. In one embodiment, the modifiedpolyhydroxylated polymers are acetal-derivatized dextran,acetal-derivatized mannan or acetal-derivatized polyvinyl alcohols.

1. Acid Degradable Linkages

As described above, the modified polyhydroxylated polymers havereversibly modified hydroxyl groups, wherein the hydroxyl groups aremodified to feature a functional group selected from the groupconsisting of acetals, aromatic acetals, ketals. In a preferredembodiment, the functional group is an acetal, aromatic acetal or aketal. In another embodiment, the modicification is made by a one-stepreaction.

In a preferred embodiment, the present acid degradable polymersdescribed herein should have a significantly lower rate of degradationin solution at pH 7.4 than at pH 5.

The modified polymers having a modified functional (e.g., acetal orketal) linkage at the modified hydroxyl groups should degrade by acidcatalyzed hydrolysis into lower molecular weight compounds that can becompletely excretable. The rate of hydrolysis of these polymers can bechanged by varying the functional group (e.g., acetal or ketal) linkagefrom slow degrading to fast degrading, the degree of modification, orthe hydrophobicity of the modification, thus providing a wide range ofrelease kinetics for drug delivery.

Thus, it is contemplated that a variety of acid degradable linkages withdifferent acid-sensitivities can be incorporated onto the polymerbackbones using this technology, allowing for excellent control of therate of polymer hydrolysis.

2. Hydrolysis of the Polymers

Drug delivery systems comprised of the polymers can be hydrolyzed torelease their contents in a pH dependent manner. In one embodiment, afeature of the present degradable polymers is the pendant modifiedhydroxyl groups on the main chain of the modified polyhydroxylatedpolymer hydrolyzes in a pH dependant manner. In a preferred embodimentthe polymers should preferably have a degradation half-life at pH 5.0 of5 minutes to 24 hours at 37° C., but a longer half life at pH 7.4 of atleast 12 hours to 250 days. In the Examples, the degradable polymershave degradation rates at pH 5.0 ranging from half-life of 5 minutes toover 26 hours.

In some embodiments, it may be useful for the polymers to have ahalf-life at pH 5.0, 37° C. of about 24 hours, and a half-life at pH7.4, 37° C. of about 250 days, in order to facilitate the slow releaseof bioactive materials. In other embodiments, it is contemplated thatthe half-life of polymer degradation at pH 5.0, 37° C. preferably be5-30 minutes, and even more preferably be less than 5 minutes and ahalf-life at pH 7.4, 37° C. of about 24 hours in order to quicklyrelease the bioactive materials.

When the modified functional groups are acetals, the acceleration of thehydrolysis kinetics of acetals from pH 7.4 to pH 5.0 is expected becausethe hydrolysis of the acetal is proportional to the hydronium ionconcentration, which should increase between pH 7.4 and pH 5.0. Thekinetics of acetal hydrolysis can be easily manipulated by introducingthe appropriate electron withdrawing or donating groups and therefore itis possible to engineer degradable polymers that have hydrolysis ratestailor-made for a given application.

A kinetic factor that may be taken into account when designing aciddegradable linkages on the modified polyhydroxylated polymer is the aciddegradable linkage's speed of hydrolysis in solution. In an embodimentwhere the goal is to hydrolyze the polymer and rapidly release thebioactive material, the acetal should preferably hydrolyze within 5-30minutes at pH 5.0 at 37° C. In one embodiment, this timescale is chosenbecause it is approximately the amount of time taken for a phagocytoseddrug delivery system to be trafficked to cellular compartments such aslysosomes. In a preferred embodiment, these particles will degraderapidly in the lysosome and cause lysosomal destabilization. Having aparticle that degrades too slowly will increase its residence time inthe lysosome and provide the lysosomal enzymes an increased chance ofhydrolyzing the bioactive material before reaching the cytoplasm throughlysosomal disruption. Therefore, in a preferred embodiment, the polymershould hydrolyze fairly rapidly at a preferred range of pH 7.4 to 4.5and even more preferably between pH 6.8 to 4.5.

In one embodiment, the present modified polyhydroxylated polymers arelargely stable at pH higher than 7.4 but hydrolyze at a pH preferablyabout 5. In one embodiment, the modified polymers are soluble in commonorganic solvents to facilitate processing into a variety of materials.In another embodiment, these modified polymers are not water soluble.

3. Methods for Polymer Modification

Generally, rate of degradation of modified polymers will depend on thedegree of modification and the hydrophobicity of the modifying group.For example, in the case of dextran modified with 2-methoxypropene, thedegradation rate of the modified polymer will depend on the amount oftime that the material is allowed to react.

For acetal modification with vinyl ethers, an example of a method can beas follows. Briefly, the polyhydroxylated polymer is dissolved in anorganic solvent such as DMSO and mixed with a vinyl ether and anacid-catalyst such as para-toluene sulfonic acid. Isolation occurs byprecipitating the material in water.

For acetal modification with acetals, an example of a method can be asfollows. The polyhydroxylated polymer is mixed with an acetal and anacid-catalyst such as para-toluene sulfonic acid over molecular sieves.After reaction, the material is isolated by precipitation into water.

For acetal modification with aldehydes or ketones, an example of amethod can be as follows. The polyhydroxylated polymer is mixed with analdehyde or ketone and an acid-catalyst such as para-toluene sulfonicacid under conditions that remove water (such as azeotropic distillationor molecular sieves). After reaction, the material is isolated byprecipitation into water.

4. Bioactive Materials

In a preferred embodiment, the invention contemplates entrapping orconjugation of such bioactive materials including but not limited to,nucleotides, oligonucleotides, polynucleotides, ribonucleotides, aminoacids, oligopeptides, polypeptides, peptoids, proteins, antigens,plasmid DNA, growth factors and hormones, interleukins,immunostimulatory agents, drugs, vaccines, neuromodulatory agents suchas neurotransmitters, stimulatory and adrenergic agents, enzymes,proteases, anticancer and antitumor agents, imaging agents, diagnosticagents, antiviral agents and antibacterial agents as well ascombinations of two or more of these species.

In specific preferred embodiments, the bioactive material is selectedfrom the group consisting of: nucleotides, oligonucleotides,polynucleotides, proteins, oligopeptides, polypeptides,immunostimulatory agents, vaccines, antigens, anti-viral agents, proteinantigens, anticancer agents and antitumor agents.

One or more of these bioactive materials can be conjugated to thepolymer chains. In one embodiment, the bioactive materials can beconjugated to the polymer through the pendant hydroxyl groups. Inanother embodiment, materials can be conjugated to the polymer throughaldehydes introduced by periodate cleavage of 1,2-diols. In the casewhere the polyhydroxylated polymers are polysaccharides, latentaldehydes are present at the reducing ends and can be used formodification. The linkage between the polymer chain and the bioactivemolecule can be designed to be cleaved under various physiologicalconditions. The bioactive material can also be adsorbed onto the surfaceof drug delivery systems, or reacted to the surface of the drug deliverysystems. The bioactive material can also be physically trapped insidethe drug delivery systems comprised of the modified polyhydroxylatedpolymers.

5. Drug Delivery Systems

In a preferred embodiment, the modified polyhydroxylated polymers aremade into particles for such applications as vaccine delivery. Typicalformulations for therapeutic agents incorporated in these deliverysystems are well known to those skilled in the art and include but arenot limited to solid particle dispersions, encapsulated agentdispersions, and emulsions, suspensions, liposomes or microparticles,wherein said liposome or microparticle comprise a homogeneous orheterogeneous mixture of the therapeutic agent. The amount of the drugthat is present in the device, and that is required to achieve atherapeutic effect, depends on many factors, such as the minimumnecessary dosage of the particular drug, the condition to be treated,the chosen location of the inserted device, the actual compoundadministered, the age, weight, and response of the individual patient,the severity of the patient's symptoms, and the like.

In one embodiment, the modified polyhydroxylated polymers made intoparticles that are 40 to 2000 nm. In general, particles can besynthesized by various techniques, such as double emulsion or spraydrying methods, as is known in the art. In one embodiment, the particlescan be made according to the procedures described by Liu, R.; Ma, G.;Meng, F.; Su, Z. J. Controlled Release 2005, 103, 31-43 and Witschi, C.;Mrsny, J. R. Pharm. Res. 1999, 16, 382-390. In a preferred embodiment,the particles are made by double emulsion, single emulsion, orprecipitation processes.

Single emulsion and the double emulsion method and precipitationprocesses can be used to produce particles from sub-micrometer tomulti-micrometer sizes; a preferable size range is from 30 nm to 5000nm, more preferably 30 nm to 2000 nm, and most preferably 40 to 200 nm.

For example, during the double emulsion method, first, the polymer isdissolved in organic solvent along with the surfactants. Then, a smallamount of aqueous solution containing the bioactive materials isdispersed into the organic/polymer phase by sonication forming a primarywater-in-oil emulsion. This primary emulsion is then dispersed into alarger amount of water containing stabilizers to form a secondarywater-in-oil-in-water emulsion. After forming the secondary emulsion,the solution is stirred until the organic phase evaporates. Whenevaporated, the polymer collapses around the aqueous bioactive materialsolution forming therapeutic-loaded particles.

In a single emulsion method, the same method is generally used as thedouble-emulsion method described above, but omitting the first emulsionstep with water.There are many nanoprecipitation techniques known tothose familiar in the art. One method would be as follows: A solution of5 mg of Ac-DEX is dissolved in 1 mL of DCM. The DCM is then addeddropwise to 10 mL of stirring water and stirred for 6 hours. Particleswere isolated by lyophilization in the presence of sucrose as acryoprotectant. (Biomaterials (2007), 869-87.)

In a preferred embodiment, the acid-degradable polymers are processed toform particles comprised of the modified polyhydroxylated polymershaving a bioactive material bound to or entrapped within the formedparticles.

In another embodiment, the modified polyhydroxylated polymers are madeinto drug delivery systems such as a small molecule implant, ortime-release device or implant. Methods and compositions useful inmaking or administering an implant or time-release device in vivo areknown and used by one having skill in the art. Examples of such methodsand compositions are described in U.S. Pat. Nos. 3,976,071; 5,876,452;7,077,859; 5,021,241, hereby incorporated by reference. For example, themodified polyhydroxylated polymers of the invention can be prepared insolid form of a needle or bar-like shape or as a bulk shaped materialand administered to the body or implanted into the body by injection oran injection-like method and whereby the bioactive material is releasedat an effective level for a long period of time after administration.

6. Loading and Loading Efficiency of Entrapped Bioactive Materials

Loading efficiency is the amount of bioactive material that is entrappedin or conjugated to within the drug delivery systems comprised of thepolymers as compared to the total starting amount of bioactive materialplaced in the loading reaction.

The loading is the amount of bioactive material contained in the polymerparticle, it is generally expressed in mass of bioactive material perunit mass of particle. The loading efficiency and the amount ofbioactive material entrapped are important aspects in light of suchfactors as the amount of bioactive material needed to be delivered tothe target for an effective dose and the amount of available bioactivematerial. A major drawback in previous therapeutics and vaccines isthere is often difficulty in obtaining large enough amounts of thetherapeutic composition of bioactive material for production. Therefore,it is a goal of the invention to make drug delivery systems with highloading capacities and efficiencies.

In one embodiment, wherein the bioactive material is a small drugmolecule for polymer-drug conjugates for applications such aschemotherapy, the degradable polymer particles should exhibit preferredloading as is known in the art. For example, the polymer particlesshould exhibit high loading efficiency to allow sufficient drugmolecules to be conjugated to the polymer or otherwise retained by thepolymer without loss of solubility of the overall formulation.

In a preferred embodiment, wherein the bioactive material loaded is DNAmaterial, the loadings and efficiencies of the drug delivery systemsshould be comparable to other microparticle systems which haveefficiencies purported to be about 1-2 μg DNA/mg polymer for 500 nm PLGAparticles. (See Garcia del Barrio, G.; Novo, F. J.; Irache, J. M.Journal of Controlled Release (2003), 86(1), 123-130). It is estimatedthat at least about 3,000-7,000 molecules of DNA can be encapsulatedwithin a single degradable polymer particle of the present invention, ifthe DNA encapsulated was 6,000 bp, which has a MW of about 4 milliondaltons. The loading efficiencies for the amount of DNA materialentrapped in degradable particles of the preferred embodiment shouldpreferably be at least 40%, more preferably at least 50% and even morepreferably at least 54%. Loadings for bacterial DNA forimmunostimulation purposes should be around 1-30 μg DNA/mg.

In a preferred embodiment, wherein the bioactive material loaded isprotein, the loading efficiencies for the amount of protein entrapped inparticles comprised of the acid degradable polymers of the preferredembodiment should be at least 20%, preferably at least 40%, morepreferably around 50%, and most preferably >90%.

7. Toxicity of Polymers and Polymer Degradation Products

Use of this invention in human and mammalian therapeutics brings upissues of the toxicity of these polymers. The viability of cells can bemeasured by the ability of mitochondria in metabolically active cells toreduce yellow tetrazolium salt (MTT) in the classical MTT assay to formformazan crystals.

In a preferred embodiment, the target cells should preferably exhibit atleast 50% viability after 24 hours of incubation with the polymers ofthe invention, more preferably at least 70% viability after 24 hours,even more preferably at least 80% viability and most preferably morethan 90% viability after 24 hours according to the MTT assay.

Polymers with high MW are not easily excreted from the body, thereforeanother aspect of the invention is to make polymers that are easily andsafely excreted by the body after being degraded in the acidicenvironments. In general it is preferred that the polymers degrade intomany small molecules and/or molecules that are non toxic and readilyexcreted from the body. The degradation products of the present modifiedpolyhydroxylated polymers of the invention should be easily excretedfrom body due to the small molecule size of the degradation productsproduced after hydrolysis of the pendant modified groups and the use ofa main chain polyhydroxylated polymer that is biocompatible (e.g., apolysaccharide such as dextran). Another aspect of the invention is tomake particles that are easily and safely excreted by the body afterbeing degraded in the acidic cellular compartment. In general it ispreferred that the particles degrade into degradation products that arelinear polymers and/or smaller molecules (e.g., 10,000 daltons or less),and that the degradation products are not toxic to a mammalian subject.

B. Applications for Modified Polyhydroxylated Polymers

This strategy for the synthesis of modified polyhydroxylated polymershas many applications including the delivery of bioactive materials,including but not limited to polynucleotides, polypeptides, proteins,peptides, organic molecules, antibodies, vaccines, antigens, geneticagents, small drugs or therapeutic agents, into the cytoplasm ofphagocytic cells, site of inflammation, tumor tissues, endosomes, orother sites of low pH. Thse materials can also be fashioned into bulkmaterials such as sutures, scaffolds, and implants.

1. Vaccine Therapeutics

In one embodiment, the polymers of the present invention would haveapplications in vaccine therapeutics and disease prevention. Proteinloaded particles prepared using these polymers could be injected into apatient, stimulating phagocytosis by macrophages and antigen presentingcells.

In one embodiment, the acid-degradable modified polyhydroxylated polymerparticles are delivered to antigen presenting cells and thenphagocytosed and trafficked to the lysosome or phagolysosome of thecells. The mild acidic conditions found in lysosomes and phagolysosomesof APCs should cause the pendant acetal groups along the polymerbackbones to be hydrolysed thereby degrading the particles. This acidhydrolysis of the acid-degradable linkage causes degradation of thepolymers.

The particles comprised of the acid degradable polymers of the inventionwould be particularly useful in combating infections that need a strongcytotoxic T lymphocyte response, including diseases such as HIV/AIDS andHepatitis C infections. Examples of such antigens which can be used asbioactive material and entrapped in the particles of the presentinvention, include but are definitely not limited to, the TAT proteinfrom HIV, the ENV protein from HIV, the Hepatitis C Core Protein fromthe Hepatitis C virus, the prostatic acid phosphatase for prostatecancer and the protein MART-1 for melanoma.

In one embodiment, the modified polyhydroxylated polymers particlesenhance CTL activation by dendritic cell (DC)-targeting. OVA isencapsulated in acid-degradable polymeric particles further conjugatedwith anti-DEC-205 mAbs monoclonal antibody. The particles are taken upby DEC-205 expressing dendritic cells in vivo. After hydrolysis in theacidic lysosome of DCs, encapsulated OVA is released into the cytoplasm.

In another embodiment, signal peptides are attached to the particle. Anysuitable signal peptide can be used in the particles of the invention.The peptide should be able to target (i.e., mediate entry andaccumulation) a particle to a subcellular compartment and/or organelleof interest. Signal peptides are typically about about 5 to about 200amino acids in length. Suitable signal peptides include, e.g., nuclearlocalization signal peptides, peroxisome-targeting signal peptides, cellmembrane-targeting signal peptides, mitochondrial-targeting signalpeptides, and endoplasmic reticulum-targeting signal peptides, andtrans-Golgi body-targeting signal peptides. Signal peptides may alsotarget the particles to any cell surface receptor including e.g.epidermal growth factor receptors (EGFR), fibroblast growth factorreceptors (FGFR), vascular endothelial cell growth factor receptor(VEGFR), integrins, chemokine receptors, platelet-derived growth factorreceptor (PDGFR), tumor growth factor receptora, and tumor necrosisfactor receptors (TNF).

Nuclear localization signal peptides typically comprise positivelycharged amino acids. Endoplasmic reticulum targeting signal peptidestypically comprise about 5 to about 10 hydrophobic amino acids.Mitochondria targeting signal peptides are typically about 5 to about 10amino acids in length and comprise a combination of hydrophobic aminoacids and postively charged amino acids. Peroxisome targeting signalpeptides include PTS1, a 3 amino acid peptide and PTS2, a 26-36 aminoacid peptide. Examples of signal peptide sequences include but are notlimited to the following sequences in Table 1.

TABLE 1 Target Source Sequence Nucleus SV-40 large  PPKKKRKVPPKKKRKV T antigen (SEQ ID NO: 1) Nucleus Tat protein  YGRKKRRQRRR  of HIV(SEQ ID NO: 2) Endoplasmic KDELA KDELA KDELA KDEL  Reticulum(SEQ ID NO: 3) Mitochondria Cytochrome  SVTTPLLLRGLTGSARRLP  C oxidaseVPRAKIHSL (SEQ ID NO: 4) Peroxisome SKLA SKLA SKLA SKLA  (SEQ ID NO: 5)Cell Membrane KLNPPDESGPCMSCKCVLS  (SEQ ID NO: 6) Cell Membrane GAP-43MLCCMRRTKQVEKNDEDQKI  (SEQ ID NO: 7)

Signal peptides can be chemically synthesized or recombinantly produced.In general, the nucleic acid sequences encoding signal peptides andrelated nucleic acid sequence homologues are cloned from cDNA andgenomic DNA libraries or isolated using amplification techniques witholigonucleotide primers. Standard techniques are used for nucleic acidand peptide synthesis, cloning, DNA and RNA isolation, amplification andpurification. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

In another embodiment, the particles are decorated with a targetingfunctional group or other cell penetrating peptides to penetratenon-phagocytic cells. For example, targeting functional groups includeantibodies, various oligopeptides, or carbohydrate moieties,Cell-penetrating peptides can also include oligopeptides such asoligomers of arginine or polymers rich in arginine motifs.

In one embodiment, immunostimulatory groups are attached to, displayedon, or encapsulated in the particle. Examples of immunostimulatorygroups include but are not limited to mannose, plasmid DNA,oligonucleotides, ligands for the Toll-like receptors, interleukins andchemokines T-cells activate B-cells to secrete Interleukin-6 (IL-6) tostimulate B cells into antibody-secreting cells.

In another embodiment, targeting antibodies are attached to theparticle. Any antibody specific for a target in vivo can be attached tothe particle to target and allow particle delivery of the bioactivematerial.

For example, in one embodiment, the acid-degradable polymers particlesenhance CTL activation by dendritic cell (DC)-targeting as describedabove.

2. Gene Therapy

In another embodiment, the polymers of the invention would be used toprepare drug delivery systems for gene therapeutics. Cationic polymerswould be especially relevant for this application because polycationscan complex with DNA. Since gene therapy involves the delivery of asequence of DNA to the nucleus of a cell, the particles comprised ofthese polymers of the invention would be especially suited for thisapplication. Once a polynucleotide is delivered by the drug deliverysystems to the cytoplasm, the polynucleotide can undergo translationinto a protein. This has the potential, then, to make proteins that arenot normally produced by a cell.

In a preferred embodiment, the bioactive material is a plasmid thatencodes for a protein or antigenic peptide initially. For example, onewould use a plasmid that encodes for a protein that would displayantigens for cancer. These proteins are not easy to generate inmulti-milligram to gram quantities to be delivered to a patient,therefore using the present particle delivery systems prepared withpolymers of the present invention to deliver plasmid DNA encoding theseantigens is a preferred alternative.

In addition to encoding for a gene, plasmid DNA has the addedcharacteristic of generating an immune response because plasmid DNA isgenerated from bacteria. Other potential bioactive materials are CpGoligonucleotides that are also derived from bacterial DNA. Bacterial DNAhas two major differences compared with vertebrate DNA: 1) bacterial DNAhas a higher frequency of CG dinucleotides in the sequence ( 1/16dinucleotides in microbial DNA are CG pairs, but only 25% of that isobserved in vertebrate DNA); and 2) bacterial DNA is unmethylated ascompared to vertebrate DNA which is often methylated. Vertebrate systemswill recognize the DNA then as being foreign, and the cell should reactas for a bacterial infection. This immune response is manifested in theproduction of cytokines and interleukins that then go on to activate Tcells, B cells, and other cells, proteins, and cellular machineryinvolved in the immune response.

3. Directing Patient Immune Response Using the Helper T-Cell Response

In a further embodiment, the plasmid DNA used as the bioactive materialwould have an added interleukin sequence. (Egan, Michael A.; Israel,Zimra R. Clinical and Applied Immunology Reviews (2002), 2(4-5),255-287.) Interleukins are secreted peptides or proteins that mediatelocal interactions between white blood cells during immune response (B.Alberts et al, Molecular Biology of the Cell, 4th ed., Garland Science,2002). Different interleukins (e.g. IL-12, IL-2) will direct the type ofimmune response that is generated. IL-6, IL-1, IL-8, IL-12, and TNF-αare secreted by infected macrophages as an immune response and IL-6serves to activate lymphocytes and increase antibody production. Thedifferentiation of helper T cells into either T_(H)1 or T_(H)2 efffectorcells determines the nature of the response. A T_(H) 1 response ischaracterized by a CTL response; a T_(H)2 response is characterized byantibody production.

It has been shown by Apostolopoulos, V.; McKenzie, I. F. C. CurrentMolecular Medicine (2001), 1(4), 469-474, that activation of the mannosereceptors on the surface of APCs leads to enhanced CTL activation. Thus,the addition of the interleukin-2 or 12 (IL-2 or IL-12) gene sequence,and its subsequent translation into an interleukin protein may allow thedirection of the type of patient immune response and amplification ofthe desired CTL response by adding or displaying immunostimulatorygroups on the surface of the particles. Such immunostimulatory groupsinclude but are not limited mannose, plasmid DNA, oligonucleotides,ligands for the Toll receptors, interleukins and chemokines T-cellsactivate B-cells to secrete Interleukin-6 (IL-6) to stimulate B cellsinto antibody-secreting cells.

4. Drug Delivery Systems and Dispersion

In a preferred embodiment, bioactive drug molecules may be temporarilyattenuated by incorporation into modified polyhydroxylated polymers forapplications such as chemotherapy. Drug molecules may be incorporatedinto the polymers covalently, where the drug molecules are attached tothe main polyhydroxylated polymer chain via labile linkages. Watersoluble polymer-drug conjugates will preferably be administeredintravenously or orally, and biologically active drug molecules will bereleased from the polymer upon cleavage of the labile polymer-druglinkages. Drug molecules may also be incorporated noncovalently byentrapment of drugs into particles or implant devices fashioned fromwater insoluble variants of the acid-degradable polymers. Waterinsoluble polymers will preferably be administered orally or will beimplanted in the body, and drug molecules will be released from thepolymer upon degradation of the polymer matrix in which the drug isentrapped or conjugated.

Drug delivery systems comprised of the invention may be suspended orstored in a conventional nontoxic vehicle, which may be solid or liquid,water, saline, or other means which is suitable for maintaining pH,encapsulation of the bioactive material for an extended period of time,sufficient dispersion or dilution of the delivery systems and theoverall viability of the delivery systems for their intended use.

Preferably the delivery systems comprised of the polymers of theinvention are stored in dry state (vacumm dried) and stored at 4° C. forseveral months. The systems may be dispersed in buffer and sonicated orvortexed for a few minutes to resuspend into solution when needed.

5. Delivery of RNAi Agents.

In a preferred embodiment, the polymers of the invention can be used toprepare delivery systems for RNA interference (RNAi) agents such assmall interfering RNA (siRNA), long double-stranded RNA (dsRNA) or shorthairpin RNA (shRNA). In one embodiment siRNA in the form of doublestranded RNA molecules less than 40 nucleotides in length can beencapsulated in polymers of the invention. Encapsulation efficiency canbe improved using cationic lipids such as DOTAP(N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N- trimethylammonium methylsulfate)or cationic polymers such as PEI (polyethyleneimine) orpoly-β-aminoesters. Materials delivering siRNA have the potential tointerfere with cellular protein production. This may be therapeuticallyrelevant for treating many different genetic or pathogenic diseases aswell as cancer.

6. Pharmaceutically Effective Delivery and Dosages

The loaded drug delivery systems of the invention can be administered byvarious suitable means to a patient, including but not limited toparenterally, by intramuscular, intravenous, intraperitoneal, orsubcutaneous injection, or by inhalation. The delivery of the systems toa patient is preferably administered by injection once but does notpreclude the necessity for multiple injections that would be required toillicit the desired response. In another embodiment, the delivery systemis an implant system, wherein the polymer is implanted into an affectedtissue, such as a tumor, and allowed to degrade and release thebioactive material. For example, water insoluble degradable polymers areimplanted in the body, and drug molecules will be released from thepolymer upon degradation of the polymer matrix in which the drug isentrapped.

The amount of delivery vehicle needed to deliver a pharmaceuticallyeffective dosage of the bioactive material will vary based on suchfactors including but not limited to, the polymer solubility, thetherapeutic loading capacity and efficiency, the toxicity levels of thepolymers, the amount and type of bioactive material needed to effect thedesired response, the subject's species, age, weight, and condition, thedisease and its severity, the mode of administration, and the like.

One skilled in the art would be able to determine the pharmaceuticallyeffective dosage. In general, the amount of bioactive material thatcould be administered by the delivery systems of the invention is from 1ng to more than 1 g quantities.

EXAMPLE 1

Synthesis and Characterization of Acid-Degradable Acetal-DerivatizedDextran

We sought to create a system with the flexibility and biocompatibilityof polyester materials, but with the additional benefit of a change inrate of payload release that is sensitive to physiologically relevantacidic conditions. We discovered a solubility switching mechanism inwhich a biocompatible, water-soluble polymer could be reversiblymodified to make it insoluble in water, but soluble in organic solvents.Materials made from the modified polymer could then be degraded underthe specific conditions that reverse the original modification. Dextran,a bacterially derived homopolysaccharide of glucose, was chosen to bemodified because of its biocompatibility, biodegradability, wideavailability, and ease of modification ((a) Hermanson, G. T.,Bioconjugate Techniques. Academic Press: San Diego, 1996 (b) Naessens,M.; Cerdobbel, A.; Soetaert, W.; Vandamme, E. J., J. Chem. Technol.Biotechnol. 2005, 80, 845-860). Acetals were chosen to modify dextrandue to their well understood and tunable pH-dependant hydrolysis rates(Fife, T. H.; Jao, L. K., J. Org. Chem. 1965, 30, 1492-&).

Dextran was rendered insoluble in water by modification of its hydroxylgroups through reaction with 2-methoxypropene under acid catalysis (FIG.4, 6). The high density of pendant acetals makes the new“acetalated-dextran” (Ac-DEX) soluble in organic solvents such asdichloromethane, ethyl acetate or acetone. Based on multi-angle lightscattering data, the molecular weight of the dextran increases uponmodification from 13 kDa to 29 kDa while the polydispersity remainsessentially constant (1.13 to 1.20), suggesting coverage of the hydroxylgroups and minimal polymer cross-linking Using a standard doubleemulsion protocol, a model hydrophilic payload, ovalbumin (OVA), wasencapsulated with a protein loading of 3.7±0.4 wt % (FIG. 6). Using asingle emulsion technique, we were able to encapsulate a modelhydrophobic drug, pyrene, with a loading of 3.6±0.5 wt %. The particleswere imaged using scanning electron microscopy (FIG. 12A) and particlesize was analyzed using dynamic light scattering. The double emulsionparticles were found to have an average diameter of 230±13 nm (FIG. 16)and the single emulsion particles had similar shapes and sizes with anaverage diameter of 258±1 nm.

Masking the hydroxyl groups of dextran as acetals not only provides ahydrophobic material that is easily processable using various emulsiontechniques, it also provides a mechanism for introducing pH-sensitivity.Under mildly acidic aqueous conditions, the pendant acetal groups areexpected to hydrolyze, thus unmasking the parent hydroxyl groups ofdextran. The complete hydrolysis of Ac-DEX should result in the releaseof acetone, methanol and water-soluble dextran. To study the degradationof Ac-DEX, empty particles were prepared and incubated underphysiological (pH 7.4) or mildly acidic conditions (pH 5.0) at 37° C.The supernatant was analyzed at various times for the presence ofreducing polysaccharides using a bicinchoninic acid based assay (Doner,L. W.; Irwin, P. L., Anal. Biochem. 1992, 202, 50-53). Ac-DEX particlesincubated in pH 7.4 buffer remained as an opaque suspension for days andessentially no soluble dextran was detected after 72 hours (FIG. 14a,c). In contrast, suspensions of Ac-DEX particles in pH 5.0 buffershowed continuous release of soluble reducing polysaccharides, becomingtransparent after 24 hours, thus suggesting full dissolution of theparticles. This pH-dependent degradation of Ac-DEX particles is furtherreflected in the release profile of a model fluorescently labeledhydrophilic payload (FIG. 17). In this experiment fluoresceinisothiocyanate (FITC) labeled dextran was released from Ac-DEX particlesmuch faster under acidic conditions than in pH 7.4 buffer. Specifically,the half-life of the release of FITC-dextran at 37° C. and pH 5.0 wasabout 10 hours compared to approximately 15 days at pH 7.4.

The degradation of empty Ac-DEX particles was also followed using¹H-NMR. A suspension of particles was incubated at 37° C. in deuteratedPBS (pH 5.5) in a flame-sealed NMR tube. The release of acetone andmethanol due to acetal hydrolysis was observed and the normalizedintegrals of these compounds were plotted as a function of time (FIG. 14c and FIG. 18). The particles first released a roughly equivalent amountof acetone and methanol, which is consistent with the rapid hydrolysisrate of pendant acyclic acetals. (Fife, T. H.; Jao, L. K., J. Org. Chem.1965, 30, 1492-&) Following this phase, acetone, but not methanolcontinued to be released from the degrading particles. This second phaseis presumably due to the slower hydrolysis rate of cyclic isopropylideneacetals, signals from which appear, then subsequently disappear as theacetals are hydrolyzed ((a) Cai, J. Q.; Davison, B. E.; Ganellin, C. R.;Thaisrivongs, S., Tetrahedron Lett. 1995, 36, 6535-6536 (b) Debost, J.L.; Gelas, J.; Horton, D.; Mols, O., Carbohydr. Res. 1984, 125,329-335). Following complete hydrolysis, the ¹H-NMR spectrum of thedegraded particles showed signals corresponding only to unmodifieddextran, acetone and methanol (FIG. 19). Based on this final spectrum,it was calculated that 73% of the available hydroxyl groups weremodified and the ratio of cyclic to acyclic acetals was estimated at1.8:1. These values were calculated using the integration of the acetoneand methanol signals compared to the integration of the anomeric proton.

We have previously shown that acid-labile polyacrylamide particlesenhance protein-based vaccine efficacy in cancer treatment by enhancingMHC class I presentation and CD8⁺ T cell activation ((a) Murthy, N.; Xu,M.; Schuck, S.; Kunisawa, J.; Shastri, N.; Frechet, J. M., Proc. Natl.Acad. Sci. U. S. A. 2003, 100, 4995-5000 (b) Standley, S. M.; Kwon, Y.J.; Murthy, N.; Kunisawa, J.; Shastri, N.; Guillaudeu, S. J.; Lau, L.;Frechet, J. M. J., Bioconjugate Chem. 2004, 15, 1281-1288). However,because the particles are prepared from acrylamide, toxicity andbiocompatibility issues might limit future clinical applications. Ac-DEXbased particles are expected to be more “bio-friendly” than our previoussystem since the byproducts are dextran (a clinically used plasmaexpander), acetone (a non-toxic, metabolic intermediate) and methanol(non-toxic in small quantities). Paine, A; Davan, A. D.; Hum. Exp.Toxico. 2001, 20, 563-568.

Thus, we present a new method for the preparation of acid-sensitive,biocompatible dextran-based materials. Ac-DEX is easily synthesized andprocessed into materials encapsulating either hydrophobic or hydrophilicpayloads. Particles made from Ac-DEX become soluble in slightly acidicenvironments, releasing their cargo. Finally, due to their favorabletoxicity profiles, these particles should find use in drug deliveryapplications demanding pH-sensitive and biocompatible materials. We arecurrently investigating the functionalization and use of these and othermodified polysaccharides in vaccine and chemotherapeutic settings. Inaddition, we believe Ac-DEX has the potential to be used as scaffolds,sutures, and other bulk materials in vivo due to its physicalproperties, biodegradability, and biocompatibility.

EXAMPLE 2

Materials and Methods for the Examples

General Procedures and Materials. All reagents were purchased fromcommercial sources and used without further purification unlessotherwise specified. Water (dd-H₂O) for buffers and particle washingsteps was purified to a resistance of 18 MΩ using a NANOpurepurification system (Barnstead, USA). When used in the presence ofacetal containing materials, dd-H₂O was rendered basic (pH 8) by theaddition of triethylamine (TEA) (approximately 0.01%). ¹H NMR spectrawere recorded at 400 MHz and ¹³C spectra were recorded at 100 MHz. Toprevent acid catalyzed hydrolysis of acetal containing compounds, CDCl₃was passed through a plug of basic alumina prior to recording NMRspectra. Multiangle light scattering (MALS) experiments were performedwith a Waters 510 pump, a 7125 Rheodyne injector, a Wyatt Optilabdifferential refractive index detector and a Wyatt DAWN-EOS MALSdetector. Absolute molecular weights determined from light scatteringdata were calculated using Astra software from Wyatt assuming aquantitative mass recovery (online method). Columns were thermostattedat 35° C. MALS experiments run with THF as a solvent were performedusing two 7.5×300 mm PLgel mixed-bed C columns with a 5 micron particlesize. MALS experiments run in aqueous conditions were performed usingdd-H₂O with 5% acetic acid as a solvent and Viscotek C-MBMMW-3078 andC-MBHMW-3078 cationic columns (7.8 mm×300 mm) in series. Fluorescencemeasurements were obtained on a Fluorolog FL3-22 spectrofluorometer(Horiba Jobin Yvon) or a Spectra Max Gemini XS (Molecular Devices, USA)for microplate-based assays. Fourier transform infrared spectroscopy(FT-IR) was carried out on a 3100 FT-IR spectrometer (Varian, USA).UV-Vis spectroscopic measurements were obtained from samples in quartzcuvettes using a Lambda 35 spectrophotometer (Perkin Elmer, USA) orusing a Spectra Max 190 (Molecular Devices, USA) for microplate-basedassays. RAW 309 and HeLa cells were obtained from ATCC (Manassas, Va.)and grown according to ATCC's directions.

EXAMPLE 3

The Acetalation of Water-Soluble Polyhydroxylated Polymers Resulting inpH-Sensitive Hydrophobic Polymers: Examples of Modification ofPolyhydroxylated Polymers

Synthesis of Acetalated Dextran (Dimethyl Acetal Dextran: Ac-DEX). Aflame-dried flask was charged with dextran (M_(w)=10 500 g/mol, 1.00 g,0.095 mmol) and purged with dry N₂. Anhydrous DMSO (10 mL) was added andthe resulting mixture was stirred until complete dissolution of thedextran was observed. Pyridiniump-toluenesulfonate (15.6 mg, 0.062 mmol)was added followed by 2-methoxypropene (3.4 mL, 37 mmol). The flask wasplaced under a positive pressure of N₂, then sealed to preventevaporation of 2-methoxypropene. After 6 h, the reaction was quenchedwith TEA (1 mL, 7 mmol) and the modified dextran was precipitated indd-H₂O (100 mL). The product was isolated by centrifugation at 4 600×gfor 10 min and the resulting pellet was washed thoroughly with dd-H₂O(2×50 mL, pH 8) by vortexing and sonication followed by centrifugationand removal of the supernatant. Residual water was removed bylyophilization, yielding “acetalated dextran” (Ac-DEX) (1.07 g) as afine white powder. IR (KBr, cm⁻¹): 3444, 2989, 2938, 1381, 1231, 1176,1053, 853. ¹H NMR (400 MHz, CDCl₃): δ 1.39 (s, br, 25H), 3.25 (br, 6H),3.45 (br, 2H), 3.60-4.15 (br, 12H), 4.92 (br, 1H), 5.13 (br, 1H).

2) Ethoxyacetal Modified Dextran

If the generation of methanol on degradation must be strictly avoided,modification using 2-ethoxypropene rather than 2-methoxypropene can becarried out. Preparation of ethoxyacetal modified dextran is carried outin the same manner as the preparation of Ac-DEX using 2-ethoxypropene inplace of 2-methoxypropene.

3) FITC-Dextran

Fluorescein isothiocyanate (FITC) modified dextran was acetalated in thesame manner as described above except FITC-dextran (M_(w)=66100 g/mol,10 mg fluorescein/g dextran) was substituted for dextran.

4) Alkyne Modified Dextran

Dextran bearing pendant alkyne groups (M_(w)=10500 g/mol, approximately6 alkyne groups per 100 glucose repeat units) was acetalated in the samemanner as described above except alkyne-modified dextran was used inplace of dextran. Alkyne-modified dextran was prepared according to theprocedure reported by De Geest et al., Chem Commun (Camb) 2008, 190-2.

5) Mannan

Mannan, a homopolysaccharide of mannose was acetalated in the samemanner as described above for dextran except mannan (M_(w)=30000-70000g/mol, from Saccharomyces cerevisiae) was used in place of dextran, thevolume of DMSO was doubled, and the reaction was quenched after 20 h.

6) Maltodextrin

Maltodextrin, a homopolysaccharide of glucose was acetalated in the samemanner as described above for dextran except maltodextrin(M_(w)=2300-4100 g/mol, from potato starch) was used in place ofdextran.

7) Polyvinyl Alcohol

Polyvinyl alcohol (PVA, M_(w)=13000-23000 g/mol, 87-89% hydrolyzed) wasacetalated in the same manner as described above for dextran except PVAwas used in place of dextran, the volume of DMSO was increased 10 fold,the amount of 2-methoxypropene was reduced to 2.5 equivalents permonomer repeat unit, and the reaction was quenched after 15 min.

8) THP Modified Dextran

A flame-dried flask was charged with dextran (M_(w)=10,500 g/mol, 300mg, 0.029 mmol) and purged with dry N₂. Anhydrous DMSO (3 mL) was addedand the resulting mixture was stirred until complete dissolution of thedextran was observed. Dihydropyran (3.4 mL, 37 mmol) was added followedby pyridiniump-toluenesulfonate (4.7 mg, 0.019 mmol). After stirringovernight the modified dextran was precipitated in dd-H₂O (100 mL, pH8). The product was isolated by centrifugation at 14800×g for 15 min andthe resulting pellet was washed with dd-H₂O (30 mL, pH 8) by vortexingand sonication followed by centrifugation and removal of thesupernatant. Residual water was removed by lyophilization to yield theproduct (357 mg) as a coarse white powder.

9) Benzylidene Acetal Modified Dextran

A flame-dried flask was charged with dextran (M_(w)=10,500 g/mol, 250mg, 0.024 mmol) and purged with dry N₂. Anhydrous DMSO (4 mL) was addedand the resulting mixture was stirred until complete dissolution of thedextran was observed. Benzaldehyde dimethyl acetal (0.35 mL, 2.3 mmol)was added followed by 5 Å molecular sieves (5 g) and p-toluenesulfonicacid monohydrate (15 mg, 0.08 mmol). After stirring for 22 h thereaction was quenched with TEA (0.15 mL, 1.1 mmol), the sieves wereremoved by filtration and the modified dextran was precipitated indd-H₂O (125 mL, pH 8). The product was isolated by centrifugation at14800×g for 15 min and the resulting pellet was washed with dd-H₂O (50mL, pH 8) by vortexing and sonication followed by centrifugation andremoval of the supernatant. Residual water was removed by lyophilizationto yield the product (249 mg) as a fine white powder.

EXAMPLE 4

Preparation of Modified Polyhydroxylated Polymer Particles

Preparation of Double Emulsion Particles Containing OVA. Microparticlescontaining ovalbumin (OVA) were made using a double emulsionwater/oil/water (w/o/w) evaporation method similar to that described byBilati et al. Yolles, S.; Leafe, T. D.; Meyer, F. J., J. Pharm. Sci.1975, 64, 115-6). Briefly, OVA (10 mg) was dissolved in phosphatebuffered saline (PBS, 137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4,50 μl). Ac-DEX (200 mg) was dissolved in CH₂Cl₂ (1 mL) and added to theOVA solution. This mixture was then emulsified by sonicating for 30 s onice using a probe sonicator (Branson Sonifier 450) with an outputsetting of 3 and a duty cycle of 10%. This primary emulsion was added toan aqueous solution of poly(vinyl alcohol) (PVA, M_(w)=13 000−23 000g/mol, 87-89% hydrolyzed) (2 mL, 3% w/w in PBS) and sonicated for anadditional 30 s on ice using the same settings. The resulting doubleemulsion was immediately poured into a second PVA solution (10 ml, 0.3%w/w in PBS) and stirred for 3 h allowing the organic solvent toevaporate. The particles were isolated by centrifugation (14 800×g, 15min) and washed with PBS (50 mL) and dd-H₂O (2×50 mL, pH 8) by vortexingand sonication followed by centrifugation and removal of thesupernatant. The washed particles were resuspended in dd-H₂O (2 mL, pH8) and lyophilized to yield a white fluffy solid (135 mg).

Preparation of Empty Double Emulsion Particles. Particles that did notcontain protein were made in the same manner as above omitting OVA.

Preparation of Empty PLGA Particles. Particles prepared frompoly(DL-lactide-co-glycolide) (PLGA, 85% lactide, 15% glycolide) weremade in the same manner as above substituting PLGA for Ac-DEX.

Preparation of Double Emulsion Particles Containing FITC-Dextran.Particles containing fluorescein isothiocyanate (FITC) labeled dextranwere made in the same manner as above substituting FITC-dextran(M_(w)=66 100 g/mol, 10 mg) for OVA.

Quantification of Encapsulated OVA. Ac-DEX particles containing OVA weresuspended at a concentration of 2 mg/mL in a 0.3 M acetate buffer (pH5.0) and incubated at 37° C. under gentle agitation for 3 d using aThermomixer R heating block (Eppendorf). After the particles had beenfully degraded, aliquots were taken and analyzed for protein contentusing the fluorescamine reagent and a microplate assay as described byLorenzen et al.(Heller, J., Ann. N. Y. Acad. Sci. 1985, 446, 51-66).Empty Ac-DEX particles were degraded in a similar fashion and used todetermine a background fluorescence level. The results were compared toa standard curve and the mass of OVA encapsulated was calculated. Theprotein loading was 3.7±0.4 wt % and the loading efficiency was 74%.

Single Emulsion Particle Preparation. Single emulsion particlesencapsulating pyrene were prepared according to a procedure adapted fromJung et al.(Rosen, H. B.; Chang, J.; Wnek, G. E.; Linhardt, R. J.;Langer, R., Biomaterials 1983, 4, 131-3). Briefly, Ac-DEX (49.9 mg) andpyrene (5.5 mg) were dissolved in CH₂Cl₂ (1 mL). This solution was addedto a PVA solution (3 mL, 1% w/w in PBS) and emulsified by sonicating for30 s on ice using a probe sonicator (Branson Sonifier 450) with anoutput setting of 5 and a duty cycle of 70%. The resulting emulsion waspoured into a second PVA solution (50 ml, 0.3% w/w in PBS) and stirredfor 4 h allowing the organic solvent to evaporate. The single emulsionparticles were isolated in the same manner as described for the doubleemulsion particles above. The washed particles were resuspended indd-H2O (2 mL, pH 8) and lyophilized to yield a white fluffy solid (38mg).

Quantification of Encapsulated Pyrene. The amount of encapsulated pyrenein single emulsion microparticles was determined by measuring pyrene'sabsorbance at 335 nm. Ac-DEX particles were weighed out in triplicateand dissolved in THF by sonicating the solutions for 2 min. Theresulting solutions were diluted and the absorbance at 335 nm wasdetermined. The loading of pyrene in the particles was calculated usingpyrene's molar absorptivity in THF as reported by Venkataramana et al.(Solbrig, C. M.; Saucier-Sawyer, J. K.; Cody, V.; Saltzman, W. M.;Hanlon, D. J., Mol. Pharm. 2007, 4, 47-57). The pyrene loading was3.6±0.5 wt % and the loading efficiency was 36%.

Scanning Electron Microscopy. Microparticles were characterized byscanning electron microscopy using a S-5000 microscope (Hitachi, Japan).Particles were suspended in dd-H₂O (pH 8) at a concentration of 1 mg/mLand the resulting dispersions were dripped onto silicon wafers. After 15min, the remaining water was wicked away using tissue paper and thesamples were allowed to air dry. The particles were then sputter coatedwith a 2 nm layer of a palladium/gold alloy and imaged. An SEM image ofsingle emulsion particles is presented in FIG. 12C.

Particle Size Analysis by Dynamic Light Scattering. Particle sizedistributions and average particle diameters were determined by dynamiclight scattering using a Nano ZS (Malvern Instruments, United Kingdom).Particles were suspended in dd-H₂O (pH 8) at a concentration of 1 mg/mLand three measurements were taken of the resulting dispersions. Sizedistribution histograms are presented in FIG. 16.

EXAMPLE 5

Characterization of Polymers and their Degradation Products

Particle Degradation: Detection of Soluble Polysaccharides via BCAassay. Empty Ac-DEX particles were suspended in triplicate at aconcentration of 2 mg/mL in either a 0.3 M acetate buffer (pH 5.0) orPBS (pH 7.4) and incubated at 37° C. under gentle agitation using aThermomixer R heating block (Eppendorf). At various time points, 120 μlaliquots were removed, centrifuged at 14 000×g for 10 min to pellet outinsoluble materials and the supernatant was stored at −20° C. Thecollected supernatant samples were analyzed for the presence of reducingpolysaccharides using a microplate reductometric bicinchoninic acidbased assay according to the manufacturer's protocol (Micro BCA ProteinAssay Kit, Pierce, USA; FIG. 2 a).(Gvili, K.; Benny, O.; Danino, D.;Machluf, M., Biopolymers 2007, 85, 379-91).

pH-Dependant Release of FITC-Dextran from Ac-DEX Particles. Thisexperiment was performed essentially in the same manner as above exceptFITC-dextran loaded particles were used instead of empty particles. Thequantity of FITC-dextran in the supernatant samples was determined bymeasuring the emission at 515 nm with an excitation of 490 nm. Theamount of FITC-dextran in each sample was calculated by fitting theemission to a calibration curve. The results of this experiment arepresented in FIG. 17.

Particle Degradation: ¹H NMR Study. Empty Ac-DEX particles (9.5 mg) anddeuterated PBS buffer (850 μL, pH 5.5) were added to an NMR tube, whichwas immediately flame sealed. An ¹H NMR spectrum was taken (initial timepoint) and the tube was placed in an oil bath heated to 37° C. Aftervarious time points additional ¹H NMR spectra were taken and theappearance of acetone, methanol, and signals assigned to the methylgroups of cyclic isopropylidene acetals (Sengupta, S.; Eavarone, D.;Capila, I.; Zhao, G. L.; Watson, N.; Kiziltepe, T.; Sasisekharan, R.,Nature 2005, 436, 568-572) was measured as a ratio of these peaks'integral to the integral of the internal standard peak(3-(trimethylsilyl) propionic-2,2,3,3,d₄ acid, sodium salt). The datawas normalized by dividing the values for acetone and the cyclic acetalsby six and the values for methanol by three. A stack plot of the NMRspectra at various time points is presented in FIG. 18 and spectrum ofthe final time point, which shows signals only from dextran, methanoland acetone is presented in FIG. 19.

Particle Degradation: Digital Photography. Empty Ac-DEX particles weresuspended at a concentration of 2 mg/mL in either a 0.3 M acetate buffer(pH 5.0) or PBS (pH 7.4) and incubated at 37° C. under gentle stirring.Digital photographs of the samples were obtained after various timepoints. The white object visible in some of the vials is a magnetic stirbar.

EXAMPLE 6

Control of Degradation Rate of Acetalated Dextran

By changing the amount of time the dextran was acetalated, thedegradation rate of the resulting polymer could be tuned over a 200-foldrange (FIG. 13).

Degradation was observed using the BCA assay. The tabulated values (inhours) are as follows.

TABLE 2 Degradation over time at pH 5 or pH 7.4. pH 5 pH 7.4  2 min 0.1327.9  5 min 0.27 75.6 10 min 1.7

60 min 16.3

360 min  22.6

1485 min  26.7

No degradation was observed after 48 hours for the slower degradingparticles at pH 7.4, so it was not possible to accurately calculate ahalf-life for them. The values in gray (col pH 7.4, 10 min-1485 min) areexpected values based on the difference in proton concentrationassociated with the different pHs

EXAMPLE 7

Class I Antigen Presentation Assays

In one embodiment, particles prepared from these acid degradableacetal-derivatized dextran polymers are designed to release theirbioactive material payload into the cytoplasm of cells upon lysosomaldestabilization. Higher loading capacity of the particles may also leadto greater antigen presentation of the encapsulated bioactive material.

The LacZ MHC Class I antigen presentation assay, as described bySanderson, S.; Shastri, N. in Inter. Immun. 1994, 6, 369-376, isperformed with degradable polymer particles made according to Example 3with the modified hydroxylated polymers of Example 2 to test theirability to deliver proteins into APCs for Class I antigen presentation.This experiment uses the LacZ B3Z hybridoma, which is a CTL thatrecognizes the peptide sequence, SIINFEKL (SEQ ID NO: 8), fromovalbumin, complexed with the MHC Class I molecule H-2K^(b). Thishybridoma produces β-galactosidase after encountering APCs that presentSIINFEKL as a Class I antigen, thus allowing Class I antigenpresentation to be quantified by measuring β-galactosidase activity.

A proper control would be to compare the amount presented by theparticles when incubated with the SIINFEKL peptide (SEQ ID NO: 8), whichis directly displayed on the antigen presenting cells and not deliveredto the cytoplasm of the cells first. In a preferred embodiment, thebioactive loading capacity and efficiency should lead to an absorbanceof that is equal to the saturation absorbance of the SIINFEKL peptide(SEQ ID NO: 8), control using the antigen presentation assay describedby Sanderson, S.; Shastri, N. in Inter. Immun. 1994, 6, 369-376.

The results of the Class I antigen presentation assay should showed thatgreater T-cell activation is seen for albumin loaded particles vs. freeprotein. APCs incubated with free ovalbumin are not able to activateCTLs, indicating that these APCs are unable to present free ovalbumin asa Class I antigen. This is presumably because ovalbumin endocytosed bythe APCs, is sequestered in lysosomes, and does not have access to theAPC cytoplasm. In contrast, APCs incubated with ovalbumin encapsulatedin the degradable particles, can efficiently activate CTLs. Ovalbuminencapsulated in the degradable particles was orders of magnitude moreefficient than free ovalbumin at inducing the activation of CTLsThus theacid degradable particles werecapable of delivering protein antigensinto APCs for Class I antigen presentation (FIG. 15).

Higher protein loading in the degradable particles is expected to leadto an increase in antigen presentation.

In order to assess the feasibility of using Ac-DEX based materials forvaccine applications, OVA-loaded Ac-DEX particles were incubated withRAW macrophages. B3Z cells, a CD8⁺ T-cell hybridoma engineered tosecrete β-galactosidase when its T-cell receptor engages anOVA₂₅₇₋₂₆₄:Kb complex, (Matsumoto, A.; Matsukawa, Y.; Suzuki, T.;Yoshino, H., J. Control Release 2005, 106, 172-80), were maintained inRPMI 1640 (Invitrogen, USA) supplemented with 10% fetal bovine serum, 2mM Glutamax, 50 mM 2-mercaptoethanol, 1 mM sodium pyruvate, 100 U/mlpenicillin and 100 mg/ml streptomycin. 1×10⁴ RAW macrophages were seededovernight in a 96 well plate and subsequently incubated withOVA-containing Ac-DEX particles or free OVA. After 6 h, the cells werewashed and 1×10⁵ B3Z cells were added to the macrophages and coculturedfor an additional 16 h. The medium was removed and 100 μL of CPRG buffer(91 mg of chlorophenol red b-D-galactopyranoside (CPRG, Roche, USA),1.25 mg of NP40 (EMD Sciences, USA), and 9 ml of 1 M MgCl₂ inl L of PBS)was added to each well. After six hours of incubation, Ac-DEX particlesincreased MHC class I presentation of the OVA-derived CD8⁺ T-cellepitope, SIINFEKL, by a factor of 16 relative to free OVA (FIG. 15)measured by the B3Z assay (Karttunen, J.; Shastri, N., Proc. Natl. Acad.Sci. U. S. A. 1991, 88, 3972-6). This drastic increase in presentationindicates that these particles may be promising materials for vaccinesagainst tumors and certain viruses, where MHC-I presentation is crucialfor the activation and proliferation of CD8⁺ T-cells. EXAMPLE 8

Examples of Encapsulation of Hydrophobic and Hydrophilic Cargoes inParticles Made from Acetalated Polymers

1) Ovalbumin Encapsulation

Microparticles containing ovalbumin (OVA) were made using a doubleemulsion water/oil/water (w/o/w) evaporation method similar to thatdescribed by Bilati et al., Pharm. Dev. Technol. 2003, 8, 1-9. Briefly,OVA (10 mg) was dissolved in phosphate buffered saline (PBS, 137 mMNaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4, 50 μl). Ac-Dex (200 mg) wasdissolved in CH₂Cl₂ (1 mL) and added to the OVA solution. This mixturewas then emulsified by sonicating for 30 s on ice using a probesonicator (Branson Sonifier 450) with an output setting of 3 and a dutycycle of 10%. This primary emulsion was added to an aqueous solution ofpoly(vinyl alcohol) (PVA, M_(w)=13000−23000 g/mol, 87-89% hydrolyzed) (2mL, 3% w/w in PBS) and sonicated for an additional 30 s on ice using thesame settings. The resulting double emulsion was immediately poured intoa second PVA solution (10 ml, 0.3% w/w in PBS) and stirred for 3 hallowing the organic solvent to evaporate. The particles were isolatedby centrifugation (14800×g, 15 min) and washed with PBS (50 mL) anddd-H₂O (2×50 mL, pH 8) by vortexing and sonication followed bycentrifugation and removal of the supernatant. The washed particles wereresuspended in dd-H₂O (2 mL, pH 8) and lyophilized to yield a whitefluffy solid (135 mg).

2) siRNA Encapsulation

Double emulsion particles containing siRNA were prepared as follows:siRNA (40 nmol) was dissolved in IDTE (pH 7.5, 50 μL). Ac-Dex (50 mg)was dissolved in CH₂Cl₂ (0.6 mL). DOTAP (400 μL of a 25 mg/mL solutionin chloroform) was added to the Ac-DEX solution (20% DOTAP). TheDOTAP/Ac-DEX solution was added to the siRNA solution. This mixture wasthen emulsified by sonicating for 30 s on ice using a probe sonicator(Branson Sonifier 450) with an output setting of 5 and a duty cycle of80%. This primary emulsion was then added to an aqueous solution ofpoly(vinyl alcohol) (PVA, M_(w)=13000-23000 g/mol, 87-89% hydrolyzed) (2mL, 3% w/w in PBS) and sonicated for an additional 30 s on ice using thesame settings. The resulting double emulsion was immediately poured intoa second PVA solution (10 mL, 0.3% w/w in PBS) and stirred for 3 hallowing the organic solvent to evaporate. The particles were isolatedby centrifugation (14800×g, 15 min) and washed with PBS (50 mL) anddd-H₂O (2×50 mL, pH 8) by vortexing and sonication followed bycentrifugation and removal of the supernatant. The washed particles wereresuspended in dd-H₂O (2 mL, pH 8) and lyophilized to yield a whitefluffy solid.

3) Simultaneous Ovalbumin and Imiquimod Encapsulation

Microparticles containing both the hydrophilic macromolecule OVA and thehydrophobic small molecule imiquimod were prepared in the same fashionas particles containing only OVA except that the CH₂Cl₂ was replacedwith CHCl₃ containing 2 mg/mL imiquimod.

4) FITC-Dextran Encapsulation

Particles containing fluorescein isothiocyanate (FITC) labeled dextranwere made in the same manner as above substituting FITC-dextran(M_(w)=66100 g/mol, 10 mg) for OVA.

5) Plasmid Encapsulation

Plasmid-loaded Microparticles were made using a modified double emulsionwater/oil/water evaporation method. Ac-DEX (40 mg) and apoly(β-aminoester) polymer (10 mg) were dissolved in ice-cold CH₂Cl₂ (1mL). Plasmid DNA encoding firefly luciferase reporter protein, gWIZLuciferase, was purchased from Aldevron/Genlantis (USA) and wasdissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.5) at aconcentration of 5 mg/mL. The plasmid solution (100 μL) was added to thepolymer solution and the mixture was emulsified by sonicating for 5 susing a Branson Sonifier 450 sonicator with a microtip probe, an outputsetting of 1, and a continuous duty cycle. This primary emulsion wasadded to an ice-cold aqueous solution of PVA (20 mL, 3% w/w in PBS) andhomogenized for 30 s at 10,000 rpm using an IKA T-25 Ultra-Turraxdigital homogenizer with an S25N-10G generator. The resulting doubleemulsion was immediately poured into a second PVA solution (40 mL, 0.3%w/w in PBS) and stirred for 2 h allowing the organic solvent toevaporate. The particles were isolated by centrifugation (3,000×g, 5min) and washed with PBS (1×20 mL) and dd-H₂O (2×20 mL, pH 8). Thewashed particles were resuspended in dd-H₂O (2 mL, pH 8) and lyophilizedto yield a white fluffy solid (34 mg). (2 mL, pH 8) and lyophilized toyield a white fluffy solid (135 mg). 6)

Preparation of Particles from Acetalated Mannan

Particles made from acetalated mannan were prepared as described abovesubstituting acetalated mannan for acetalated dextran and omitting theOVA solution and the first emulsion step (see FIG. 10).

7) Pyrene Encapsulation

Single emulsion particles encapsulating pyrene were prepared accordingto a procedure adapted from Jung et al. (Jung, J.; Lee, I. H.; Lee, E.;Park, J.; Jon, S., Biomacromolecules 2007, 8, 3401-3407). Briefly,Ac-Dex (49.9 mg) and pyrene (5.5 mg) were dissolved in CH₂Cl₂ (1 mL).This solution was added to a PVA solution (3 mL, 1% w/w in PBS) andemulsified by sonicating for 30 s on ice using a probe sonicator(Branson Sonifier 450) with an output setting of 5 and a duty cycle of70%. The resulting emulsion was poured into a second PVA solution (50mL, 0.3% w/w in PBS) and stirred for 4 h allowing the organic solvent toevaporate. The single emulsion particles were isolated in the samemanner as described for the double emulsion particles above.

8) Imiquimod Encapsulation

Single emulsion particles encapsulating imiquimod were preparedaccording to the same procedure used to encapsulate pyrene, but CHCl₃was used in place of CH₂Cl₂ and imiquimod was used in place of pyrene.The single emulsion particles were isolated in the same manner asdescribed for the double emulsion particles above.

9) Doxorubicin Encapsulation

Single emulsion particles encapsulating doxorubicin (Dox) were preparedaccording to a procedure adapted from Tewes et al. (Tewes, F.; Munnier,E.; Antoon, B.; Ngaboni Okassa, L.; Cohen-Jonathan, S.; Marchais, H.;Douziech-Eyrolles, L.; Souce, M.; Dubois, P.; Chourpa, I., Eur J PharmBiopharm 2007, 66, 488-92). Briefly, Dox (1 mg) was dissolved in asodium borate buffer (2m1, 50 mM, pH 8.8) and mixed extensivelyovernight with CH₂Cl₂. The CH₂Cl₂ was then isolated and evaporated to afinal volume of 1 mL. A solution of Ac-Dex (100 mg) in CH₂Cl₂ (1 mL) wasadded to the Dox. The resulting solution was added to a PVA solution (4mL, 3% w/w in PBS) and emulsified by sonicating for 60 cycles on iceusing a probe sonicator (Branson Sonifier 450) with an output setting of4 and a duty cycle of 10%. The resulting emulsion was poured into asecond PVA solution (10 mL, 0.3% w/w in PBS) and stirred for 4 hallowing the organic solvent to evaporate. The resulting particles wereisolated in the same manner as described for the double emulsionparticles above.

10) Encapsulation of Camptothecin by Nanoprecipitation

Ac-DEX particles can encapsulate organic molecules usingnoprecipitation. Camptothecin (2.4 mg) was dissolved in hot DMF (400 μL)then diluted with acetone (600 μL) containing Ac-DEX (100 mg). Thissolution was added dropwise to H₂O (10 mL, pH 8). Particles wereisolated by concentration using Centricon spin filters. Dry statestorage could be obtained by lyophilization in the presence of 10%sucrose as a cryoprotectant.

EXAMPLE 9

Toxicity of Degradable Particles.

The toxicity of bioactive material loaded degradable particles can bemeasured with the yellow tetrazolium salt,3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), assayusing RAW 309.CR1 macrophage cells (ATCC No. TIB-69, American TypeCulture Collection, Manassas, Va.). The cells are incubated with thedegradable particles in DMEM media with 10% F.B.S. The degradableparticles are aspirated from the cells and then washed several timeswith PBS and allowed to grow for 24-48 hours. The cell viability isdetermined by measuring the absorbance of the reduced MTT reagent usingthe protocol described in Freshney et al. (Freshney, I. R. (1994)Culture of animal cells, Wiley-Liss, Inc, New York, N.Y.) as compared toa control. MTT (yellow) is reduced metabolically by healthy cells inpart by the action of dehydrogenase enzymes in mitochondria, to generatepurple formazan crystals, which are solubilized by the addition of adetergent and the absorbance is measured at 570 nm. Thus, themeasurement of the ability of cells to reduce the MTT reagentmetabolically is a measurement of the health of the cell population.

RAW 309.CR1 macrophage cells are split at 5×10⁴ cells per well in a 96well plate and allowed to grow overnight. The cells are then incubatedwith the degradable particles with variable amounts of loaded ovalbuminfor 24 hours in DMEM media with 10% F.B.S. The degradable particles arethen aspirated from the cells, and then washed several times with PBSand allowed to grow for another 24 hours.

The cell viability is determined by measuring the absorbance of thereduced MTT reagent. The MTT assay is performed using 0.5, 1, 2.5 and 5mg particles/mL serum in each well with a particle loading of 10micrograms protein/mg particle. After 24 hours, number of viable cellsremaining is observed. It is preferred that at least 50%, morepreferably 80-90% viable cells remain. Thus, it can be found whether thedegradable particles of the invention are not toxic to mammalian cellsif more than 50% of the cells remain viable.For cell viabilityexperiments, degradation products of empty Ac-DEX particles were testedusing RAW macrophages (FIG. 20 b). Additionally, empty Ac-DEX particlesand empty PLGA particles were incubated with RAW macrophages (FIG. 20a). The degradation products were obtained by incubating Ac-DEXparticles in a 0.3 M acetate buffer (pH 5.0) at 37° C. under gentleagitation for 3 d. The resulting solution was desalted using a Microcon3 centrifugation filter (Millipore, USA) and lyophilized. During thedesalting and lyophilization steps the methanol and acetone releasedduring degradation was removed. Before use in the viability experiment,the lyophilized degradation products were dissolved in medium, andmethanol and acetone were added corresponding to the maximum amount ofthese byproducts released, as found in the ¹H-NMR degradation studydescribed above.

For each viability experiment, 1×10⁴ RAW macrophages were seeded in a 96well plate and allowed to grow overnight. Serial dilutions of thedegradation products, empty Ac-DEX or PLGA particles were added to thecells, which were then incubated overnight. The next morning, viabilitywas measured using the MTT assay (described above). Results arepresented as the mean of triplicate cultures±95% confidence intervals

Although toxic in large quantities, the level of methanol released byAc-DEX materials (˜7 wt. %) would be below the EPA recommended limit ofdaily exposure as long as the dosage does not exceed 7 g/kg/day. Ifcomplete elimination of methanol becomes necessary, 2-ethoxypropeneinstead of 2-methoxypropene could be used.

To assess the biocompatibility of Ac-DEX particles, we compared them toparticles prepared from an FDA approved polymer, poly(lactic-co-glycolic acid). We found no significant difference intoxicity between the two materials in both HeLa cells and RAWmacrophages. The degradation products of Ac-DEX particles were alsofound to be non-toxic (FIG. 20).

EXAMPLE 10

Release of Bioactive Material from Degradable Polymer Particles

Degradable particles are made according to Example 4 with the degradablepolymers in Example 1, encapsulating fluorescently labeled dextran andfed to macrophage cells and compared to non-degradable controlparticles. When a nondegradable particle is used, the fluorescence ismore localized showing that when nondegradable particles have been takenup by the cells, they remain sequestered in the lysosome without amechanism of release. When the acid degradable particles are used, thefluorescence is more diffuse within the cytoplasm of cells, which isindicative of cytoplasmic release of the degradable particle contents.

EXAMPLE 11

In Vivo Studies to Assess the Degradable Polymer Particle Delivery ofVaccine Therapeutics to Antigen Presenting Cells

To assess the ability of the acid-degradable protein-loaded particles todeliver protein to the cytoplasm of APCs and activate CTLs and provide aprotective immunity in vivo, a preliminary tumor protection experimentis performed using the EG7 tumor model (El-Shami, K., Tirosh, B.,Bar-Haim, E., Carmon, L., Vadai, E., Fridkin, M., Feldman, M., andEisenbach, L. (1999) MHC class I-restricted epitope spreading in thecontext of tumor rejection following vaccination with a singleimmunodominant CTL epitope. Eur. J. Immunol. 29, 3295-3301; Kim, T. S.,Chung, S. W., and Hwang, S. Y. (2000) Augmentation of antitumor immunityby genetically engineered fibroblast cells to express both B7.1 andinterleukin-7 Vaccine 18, 2886-2894). EG7 is a derivative of the thymomaEL4, which was transfected with the ovalbumin gene, making it a targetcell for CTLs activated against ovalbumin. The modified polyhydroxylatedpolymer chosen for in vivo study should demonstrate good dispersibilitywhich may be an important consideration for the study if the particlesmust be suspended in saline and injected into animals. Certain modifiedpolyhydroxylated polymers may be somewhat more difficult to suspend,most likely due to a degree of particle aggregation as a result ofhigher ovalbumin content.

Briefly, in an in vivo study, the modified polyhydroxylated particlesare injected into the foot pad of CD4 or CD8 transgenic mice to showthat the particles can activate cytotoxic T lymphocytes in vivo. Morepreferably, delivery is by injection of 50 pl of resuspended particleusing a 25 gauge syringe in the flanks of these transgenic mice. Atleast 50 μg of OVA/mouse should suffice per injection with at least 3mice per group injected. Also 150 μg of particles with OVA and a similaramount of particles used for control are injected. The lymph nodes areisolated 7 days after the injection and analyzed for antigen specific Tcell priming.

In Vivo Tumor Protection Experiment. Experiments are performed withfemale C57BL/6 mice and all immunizations are administered bysubcutaneous injections using 26 gauge needles. There are three groups(15 mice per group): control mice injected with saline (200 μL); miceinjected with free ovalbumin in saline (50 μg in 200 μL); and miceinjected with ovalbumin encapsulated in particles dispersed in saline(1.13 mg particles, corresponds to 50 μg ovalbumin, in 200 μL). A secondidentical immunization is delivered 2 weeks after the first. Then 10days after the second immunization, tumors are established byadministering an injection of 1×10⁶ EG7-OVA tumor cells in 100 μL salineinto the shaved left flank of each mouse. One week prior to injection,the EG7 cells are stained with the anti-SIINFEKL/K^(b) monoclonalantibody 25.D1-16 and the secondary goat anti-mouse antibody labeledwith R-phycoethrin (PE). Then highly ovalbumin-expressing cells arecollected using flourescence-activated cell sorting (FACS) andproliferated. After injection, the tumor growth is monitored bymeasuring two perpendicular axes using digital calipers. Tumor volume isthen calculated using the equation, volume=0.5×length x width, with thelength being the longest diameter and the width being the shortestdiameter of the two perpendicular measurements. Once the tumor reaches1.5 cm in average diameter, the mouse is removed from the experiment andeuthanized according to guidelines set by the UC-Berkeley Animal Careand Use Committee. Mice are also removed if they showed other signs ofpain or distress such as a lack of cleaning, eating, or mobility. A logrank test is used to determine p-values. A p-value of 0.05 or less isconsidered to be statistically significant.

In a previous experiment described in Standley et al., Acid-degradableparticles for protein-based vaccines: enhanced survival rate fortumor-challenged mice using ovalbumin model, Bioconjug Chem. 2004November-December; 15(6):1281-8, using polyacrylamide particles madewith an acid-degradable crosslinker, mice injected showed slower tumorgrowth and 100% survival rate after 17 days. These encouragingpreliminary results suggest that modified polyhydroxylated particlesshould stimulate an immune response against EG7 tumor cells and providea protective immune response against tumor cells in vivo.

Future experiments can seek to take advantage of the syntheticflexibility of the presently described delivery system throughincorporation of APC targeting and immunostimulatory groups such asmannose and CpG DNA into the present modified polyhydroxylatedparticles. Additional studies will also test samples with varied dosesand protein loadings to determine if these variables have the sameeffect in vivo as they do in vitro.

EXAMPLE 12

Tumor Immunotherapy with Model System

In one embodiment, Ac-DEX particles will encapsulate OVA similar to(Example 9 with B3Z assay) and be used in a tumor prophylactic andtreatment vaccination against cancer. MO5 is a B16 tumor cell line thatexpresses OVA (Falo et al. 1995, Targeting antigen into the phagocyticpathway in vivo induces protective tumour immunity. Nat Med 1:649-53).Mice are immunized subcutaneously with 2 mg of Ac-DEX particles with a2.5 wt % loading of OVA (50 μg of OVA) in the left flank on day 0. Othergroups include mice which are injected with either OVA, saline, orAc-DEX particles encapsulating OVA and an immunostimulatory CpGstimulant. We have shown earlier that adding CpG with acid labileparticles drastically increases the efficacy of treatment(Standley etal. 2007, Incorporation of CpG oligonucleotide ligand intoprotein-loaded particle vaccines promotes antigen-specific CD8 T-cellimmunity. Bioconjug Chem 18:77-83). CpG can be either co-injected withthe particles, or it can be encapsulated inside the particles. On day 7mice are challenged with 200,000 MO5 tumor cells that are implantedsubcutaneously in the right flank

After injection, the tumor growth is monitored by measuring twoperpendicular axes using calipers. Tumor volume is then calculated usingthe equation, volume=0.5×length x width, with the length being thelongest diameter and the width being the shortest diameter of the twoperpendicular measurements. Once the tumor reaches 1.5 cm in averagediameter, the mouse is removed from the experiment and euthanizedaccording to guidelines set by the UC-Berkeley Animal Care and UseCommittee. Mice are also removed if they show other signs of pain ordistress such as a lack of cleaning, eating, or mobility. Mice injectedwith PBS and nonencapsulated OVA have the fastest tumor onset and tumorgrowth. Mice injected with OVA encapsulated in Ac-DEX particles have adelay in cancer onset and increase in survival time. Mice receivingencapsulated OVA and coencapsulated or coinjected CpG have the slowesttumor onset and the longest survival time.

After tumor prophylactic experiments, treatment experiments areperformed. Mice are implanted with 200,000 MO5 tumor cells on day 0.After day 0, mice are immunized on day 6 with either saline, 50micrograms of OVA, OVA encapsulated in Ac-DEX particles, or OVAencapsulated in Ac-DEX particles with coencapsulated or coinjected CpG.After injection mice are monitored for tumor growth. Tumors shrink anddisappear after a coinjection of OVA and CpG in Ac-DEX particles.

EXAMPLE 13

Tumor Immunotherapy with Clinically Relevant System

In another embodiment, Ac-DEX particles will encapsulate naturalantigens inherent in the tumors against which are vaccinated. Forinstance, the mouse B16 tumor possess the antigen tyrosinase-relatedprotein (TRP2), which is also present in many human melanoma tumors(Jerome et al. 2006, Cytotoxic T lymphocytes responding to low dose TRP2antigen are induced against B16 melanoma by liposome-encapsulated TRP2peptide and CpG DNA adjuvant. J Immunother 29:294-305). The recombinantform of TRP2 will be encapsulated in Ac-DEX particles similar to theexperiments done with OVA. On day 0, mice are immunized in the leftflank with 2 mg of particles that are 2.5 wt % (50 μg of TRP2). As withthe OVA tumor experiment, other groups include immunization with saline,free TRP2, or TRP2 particles with coencapsulated or coinjected CpG.After vaccination, on day 7, 200,000 B16 tumor cells are implanted inthe right flank and mice are monitored for tumor growth. Mice receivingencapsulated OVA and coencapsulated or coinjected CpG have the slowesttumor onset and the longest survival time.

After tumor prophylactic experiments, treatment experiments areperformed. Mice are implanted with 200,000 MO5 tumor cells on day 0.After day 0, mice are immunized on day 6 with either saline, 50micrograms of TRP2, TRP2 encapsulated in Ac-DEX particles, or TRP2encapsulated in Ac-DEX particles and CpG. After injection mice aremonitored for tumor growth. Tumors shrink and disappear after acoinjection of OVA and CpG in Ac-DEX particles.

Besides injection with recombinant TRP2, known immunodominant peptidesequences of TRP2, approximately seven to nine amino acids long, can beincorporated inside the Ac-DEX particles. Methods of incorporating thepeptide inside the particles could be as simple as a double emulsiontechnique (Batanero et al. 2002, Biodegradable poly(DL-lactideglycolide) microparticles as a vehicle for allergen-specific vaccines: astudy performed with Ole e 1, the main allergen of olive pollen. JImmunol Methods 259:87-94). Other methods familiar to those in the artcan be used in incorporating small peptides inside the particles.

EXAMPLE 14

Ac-DEX Particles for Delivery of Chemotherapeutic Agents

In one embodiment, Ac-DEX particles will encapsulate chemotherapeuticagents such as doxorubicin (DOX) inside the particles by using themethod described above. In order for the particles to have a longcirculation time in vivo, particles can be coated with polyethyleneglycol (PEG). It is well known to those in the art that particles coatedwith PEG have increased blood circulation in vivo (Shenoy et al. 2005,Poly(ethylene oxide)-modified poly(beta-amino ester) nanoparticles as apH-sensitive system for tumor-targeted delivery of hydrophobic drugs:part 2. In vivo distribution and tumor localization studies. Pharm Res22:2107-14). We plan on coating particles with a Pluronic triblockpolymer in which the polypropylene glycol unit is absorbed onto theAc-DEX particles leaving the PEG chains free to cover the surface of theparticles. If a higher pegylation is desired, the alkyne dextranparticles described earlier could be conjugated to azide functionalizedPEG chains.

To determine the maximum in vivo tolerated dose for the particles, avarying concentration of doses of 0 (PBS), 20, 40, and 60 mg/kg DOXequivalents are injected intravenously. The weights and general healthof the mice are monitored until the ninth day after injection. Based onweight loss, the maximum injection of particles tolerated in mice iscalculated. Once determined, mice are implanted subcutaneously with aB16 tumor. After 3 days mice are injected with PBS, free DOX, Ac-DEXparticles encapsulating DOX, pegylated-Ac-DEX particles, or Doxcil (acommercially available doxorubicin/liposome conjugate). Mice aremonitored for tumor growth. Once the tumor reaches an average diameterof 1.5 cm, the mice are sacrificed. Mice injected with PBS have thefastest tumor growth followed by, free Dox, Ac-DEX particles, Doxcil andfinally PEGylated Ac-DEX particles.

Further experiments could be done encapsulating multiplechemotherapeutic drugs such as camptothecin, paxlitaxel, and cisplatninin a single carrier construct.

EXAMPLE 15

Bioengineering Scaffolding and Sutures

In another embodiment Ac-DEX polymer is used as a bioengineeringscaffold. There are many methods in making tissue engineering scaffoldsout of biodegradable polymers such as poly(lactic-co-glycolic acid)(PLGA). PLGA has been used to make sutures, macroscopic implantabledisks, and tissue scaffolds. Since Ac-DEX particles are soluble in mostof the organic solvents PLGA is soluble in, Ac-DEX can be used toreplace PLGA in these applications that are known by those in the art.Alternatives for PLGA are needed because in certain in vivoapplications, PLGA has been known to lower the local pH in and aroundthe polymer (Shenderova et al. 1999, The acidic microclimate inpoly(lactide-co-glycolide) microspheres stabilizes camptothecins. PharmRes 16:241-8). One method in particular is the gas foaming/leachingprocess (Ennett et al. 2006, Temporally regulated delivery of VEGF invitro and in vivo. J Biomed Mater Res A 79:176-84). Ac-DEX particles arecombined with NaCl particles (diameter, 250-425 nm) and 1% (w/v)alginate solution. The alginate serves to increase protein incorporationand functions as a stabilizer. This mixture is lyophilized and pressedinto a pellet using a Carver press. The scaffolds will then be placedunder high pressure CO2 gas and allowed to equilibrate. The pressurewill be rapidly returned to ambient conditions leading to athermodynamic instability and causing the polymer to foam and create aninterconnected structure around the NaCl. Both types of particles foamand fuse together to create the scaffold, and no distinct particles ormicrospheres are present in the scaffold after this processing. The NaClis leached in a CaCl2 solution to create a macroporous structure.

Bioengineering scaffolds can be used to encapsulate growth factors forbones, blood vessles, cardiovascular tissue, nerve cells and othertissue systems. Growth factors such as vascular endothelial growthfactor (VEGF) can be incorporated in the Ac-DEX particles. Incorporationof VEGF inside the scaffold can increase blood vascular growth and anincrease in vascularization (Sun et al. 2005, Sustained vascularendothelial growth factor delivery enhances angiogenesis and perfusionin ischemic hind limb. Pharm Res 22:1110-6).

Another application where Ac-DEX may be an advantageous replacement forPLGA is absorbable sutures. Sutures are prepared using methods known tothose familiar with the art. Ac-DEX sutures are found to have tensilestrength that is comparable to that of PLGA closure and sufficientflexibility for use. Rate of absorption is adjusted according to theneeds of the wound closure. Generally, Ac-DEX sutures are appropriatefor situations where absorption must occur faster than occurs with PLGAsutures or in situations where it is desirable to avoid localacidification from PLGA degradation.

EXAMPLE 16

Gene Delivery Using Modified Hydroxylated Polymers

An embodiment for the use of Ac-DEX polymer, is the encapsulation of DNAinside Ac-DEX particles. Plasmid DNA was encapsulated into Ac-DEX usinga double emulsion technique (Gwak and Kim, 2008, Poly(lactic-co-glycolicacid) nanosphere as a vehicle for gene delivery to human cordblood-derived mesenchymal stem cells: comparison with polyethylenimine.Biotechnol Lett). Briefly, 1 ml of luciferase plasmid DNA (pCMV-Luc, 2mg/ml) in Tris/EDTA buffer was emulsified in 2 ml Ac-DEX solution (5%w/v in methylene chloride) with a probe sonicator for 1 min. Thewater-in-oil emulsion was further emulsified in 25 ml of a 2% (w/v) PBSbuffered aqueous solution of polyvinyl alcohol using a homogenizer foran additional minute. The emulsion was stirred for 8 h at roomtemperature to remove the methylene chloride. The nanospheres wererecovered by centrifugation at 12,000 g for 15 min at 4° C. Theremaining pellet was resuspended in distilled water (with one drop oftriethylamide) by vortexing and washed five times with distilled waterto remove PVA and unentrapped agent. The washings were performed bycentrifugation at 12,000 g for 15 min at 4° C. After washing, particleswere lyophilized.

Particles were compared to Polyethylenimine (PEI), a polycationicpolymer used commonly in transfecting mammalian cells with DNA. Thefirst experiment was to compare the toxicity of Ac-DEX particles to PEIpolymers. HeLa cells were plated at 40000 cells/well in 96 well plates.Varying concentrations of PEI or Ac-DEX particles are cultured withcells for 4, 8, or 24 hours. After these time points cell viability wasmeasured. HeLa cells grown with Ac-DEX were more viable compared to thecells grown with PEI.

Next, Ac-DEX particles encapsulating a luciferase plasmid were testedfor transfection efficiency. Cells were incubated with particles at 5μg/ml of DNA. Luciferase activity was looked at day 1, 2, 5, 10 and 20days. Cells were then lysed, proteins were purified and luciferaseactivity was measured. Ac-DEX particles had somewhat lower activity thanthe PEI control, but because Ac-DEX particles are less toxic, they are agood candidate for gene delivery.

EXAMPLE 17

Increased Uptake Using Cell Penetrating Peptides

Cell penetrating peptides (CPP) were used to increase uptake ofparticles inside non-phagocytic cells. Ac-DEX particles were suspendedin PBS at a concentration of 1 mg/mL and aliquots (1.5 mL) weretransferred to microcentrifuge tubes. The samples were centrifuged(10,000×g, 15 min) and the supernatant was removed. Using a bathsonicator, the particles were resuspended in a solution (1.5 mL) ofaminooxyacetyl-K-(R)₉-COOH (10 mg/mL in PBS, pH 7.4). After 2 days atroom temperature under gentle agitation, these particles were washedthoroughly. Particles encapsulating plasmid and functionalized with CPPwere prepared in the same manner, except the reaction was only allowedto proceed for 1 d. This reaction leads to the formation of oximelinkages between the aminoxy group and the latent aldehydes at thereducing ends of the modified polysaccharide. The use of hydrazides orhydrazines can similarly lead to the formation of acid-labile hydrazonelinkages. Additionally, reaction with amines followed by reduction withNaBH₃CN or similar reducing agents can lead to amine linkages throughreductive amination. .

The toxicity of Ac-DEX particles with surface CPPs was tested. HeLacells were seeded at 40 000 cells per 96 well plate. Two-fold dilutionsof particles starting at 1,000 μg/ml with or without CPP on the surfacewere cultured with HeLa cells. After 24 hours, cells were washed toremove any nonendocytosed particles and allowed to rest for anadditional 24 hours. Then a MTT assay was done to measure cellviability. Ac-DEX particles with and without CPP on the surface were nottoxic up to 1 000 microgram/ml.

Particles with CPP on the surface were tested for transfectionefficiency. Using the method described above, a luciferase plasmid wasincorporated inside the particles and tested for efficacy with HeLacells. CPP on the surface of the Ac-DEX particles statisticallyincreased the level of transfection compared to normal Ac-DEX particles.

EXAMPLE 18

Chemotherapy for Restenosis

Restenosis typically occurs after angioplasty is done on blood vessles.Restenosis in part is due to an inflammatory response around the sitewhere the angioplasty occurred. Restenosis can be prevented by thetreatment with rapamycin and dexamethasone (Zweers et al. (2006) Releaseof anti-restenosis drugs from poly(ethyleneoxide)-poly(DL-lactic-co-glycolic acid) nanoparticles. J Control Release114:317-24). Ac-DEX particles can encapsulate both rapamycin anddexamethasone using single emulsion techniques or salting methods.Particles can be pegylated as earlier stated.

A HPLC C18 column is used to study the drug loading of rapamycin insidethe Ac-DEX particles. Particles are dissolved with acetonitrile, andthen injected into the HPLC. Based on a standard curve, the rapamycincontent is calculated. While the dexamethasone content is calculated bydissolving approximately 5 mg of Ac-DEX particles in deuterated DMSO andcomparing the integral of the dexamethasone peak to the anomeric peak ondextran.

During restenosis, there is an uncontrolled proliferation of smoothmuscle cells (SMCs). To test the efficacy of the particles in vitro,smooth muscle cells were enzymatically isolated from the thoracic aortasof adult male Sprague-Dawley rats (6-7 weeks old, approximately 150 g).Cells were plated into 96-well flat-bottom tissue culture plates at afinal concentration of 20,000 cells/ml. SMCs were incubated at 37° C. in5% humidified CO₂ with Ac-DEX rapamycin loaded particles at variousconcentrations. Cell proliferation was induced by mitogenic stimulationwith rat platelet-derived growth factor (PDGF, 50 ng/ml). SMC werequantified after 4 days using a MTT assay. Particles loaded withrapamycin have a similar activity compared to nonencapsulated rapamycin.

After in vitro tests, in vivo tests will be done by methods known tothose familiar in the art.

EXAMPLE 19

Drug-Eluting Stents

As described in Example 18, restenosis (an exaggerated neointimalproliferative response) can be triggered in a number of patientsfollowing the placement of coronary stents as a treatment forobstructive cardiovascular disease. In order to prevent restenosis, anumber of drug-eluting stents have been used (i.e., Randade et al.(2004) Physical characterization of controlled release of paclitaxelfrom the TAXUS™ Express²™ drug-eluting stent. Journal of BiomedicalMaterials Research Part A 71A:625-634) Restenosis is a result of anumber of processes occurring both acutely (i.e., the inflammatoryresponse caused by mechanical injury to the arterial wall secondary toballoon dilation and stent deployment) as well as in theintermediate/long-term (proliferation of SMCs). Due to the multipleprocesses involved in restenosis, which occur after various periods oftime, it is beneficial to control the release rate of drugs fromdrug-eluting stents (see Venkatraman et al. (2007) Release profiles indrug-eluting stents: Issues and uncertainties. Journal of ControlledRelease 120:149-160). Therefore materials such as Ac-DEX, which haveeasily controlled degradation rates should be promising materials forstent coatings or for the production of fully biodegradable stents.

Using methods known to those familiar in the art, Ac-DEX or otheracetal-modified polyhydroxylated polymers can be used to coat metalstents with formulations containing various concentrations ofpaclitaxel, rapamycin (sirolimus), dexamethasone or any mixture of thesedrugs. Alternatively, these drugs can be encapsulated in microparticlesusing techniques such as emulsion/solvent evaporation ornanoprecipitation and the microparticles can be used to coat stents.Additionally, fully biodegradable stents can be made from Ac-DEX usingmethods known to those familiar in the art. Based on the degree and typeof acetal modification, the release rates of drugs from the stentsdescribed above can be varied. The drug release profiles from the stentsdescribed above are determined by incubating individual stents in mediumat 37° C. and pH 7.4. The medium is removed after various time pointsand analyzed using high performance liquid chromatography (HPLC) todetermine the amount of each drug released.

The drug-eluting stents prepared above using Ac-DEX or otheracetal-modified polyhydroxylated polymers can be tested in vitro fortheir ability to reduce proliferation of SMCs using theantiproliferation assay described in Example 18. Following in vitrotests, the stents will be evaluated for their in vivo efficacy usingmethods known to those familiar in the art.

EXAMPLE 20

Knockdown of Protein Expression Using Encapsulated siRNA

HeLa-luc cells were seeded (˜15,000 cells/well) into each well of a96-well clear tissue culture plate and allowed to attach overnight ingrowth medium. Growth medium was composed of DMEM (with phenol red), 10%FBS, and 1% glutamine. Particle samples encapsulating siRNA wereprepared at 1000 μg/mL in medium (without antibiotics) by alternatelyvortexing and sonicating in a Branson 2510 water bath for 20 s togenerate homogeneous suspensions. The samples were then serially dilutedin medium to give the indicated particle concentrations. Existing mediumwas replaced with 100 μl of each particle dilution (or medium only) intriplicate wells of each 96-well plate. The cells were allowed to growfor an additional 48 h before being analyzed for gene expression.Lipofectamine 2000 was used as a positive control for siRNA delivery andwas prepared according to the manufacturer's instructions. In addition,complexes of DOTAP and siRNA were prepared by mixing DOTAP and siRNAsolutions and incubating for 30 min prior to adding to the cells.

The cells of one of the plates were washed with PBS (containing Mg2+ andCa2+, 3×100 μL), GLO LYSIS Buffer (120 μL, Promega, USA) was added toeach well and the plate was incubated at rt. After 20 min, 100 μL fromeach well was transferred to the wells of a white 96-well tissue cultureplate. STEADY-GLO luciferase assay reagent (Promega) was reconstitutedaccording to the manufacturer's instructions and added to each well (100μL) using an automatic injector. The plate was read using a GLOMAX 96microplate luminometer (Promega) with a 2 s integration rate.

To determine the total protein content in each well, the cells of asecond clear plate were washed as above and lysed with M-PER mammalianprotein extraction reagent (50 μl, Pierce-Thermo Fisher, USA) for 20 minat rt. PBS (50 μl) was added to each well, and the plate was brieflyvortexed to mix. Samples from each well (50 μl) were added to the wellsof a black 96-well tissue culture plate (Corning) already containing PBS(100 μl per well). A solution of 3 mg/mL fluorescamine in acetone (50μl) was added to each well, and the plate was briefly vortexed to mix.After 5 min, the fluorescence in each well was measured using aplate-reading fluorimeter (excitation=400 nm, emission=460 nm). Proteinconcentrations were determined using BSA as a standard.

Significant dose dependent knockdown of luciferase production relativeto total protein production was found to be caused by siRNA encapsulatedin particles but not by free siRNA.

The present examples, methods, procedures, specific compounds andmolecules are meant to exemplify and illustrate the invention and shouldin no way be seen as limiting the scope of the invention. Any patents,publications, publicly available sequences mentioned in thisspecification are indicative of levels of those skilled in the art towhich the invention pertains and are hereby incorporated by referencefor all purposes to the same extent as if each was specifically andindividually incorporated by reference.

1. A composition comprising a modified polyhydroxylated polymercomprising a polyhydroxylated polymer having reversibly modifiedhydroxyl groups.
 2. The composition of claim 1, wherein at least 20%,25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100%of the hydroxyl groups in the polymer are modified.
 3. The compositionof claim 2, wherein the hydroxyl groups are modified by a one-stepreaction to feature a functional group.
 4. The composition of claim 3,wherein the functional group is selected from the group consisting ofacetals, aromatic acetals, and ketals.
 5. The composition of claim 1,wherein said polymers are processed to form particles, sutures, bulkmaterials, tissue engineering scaffolds, or implants.
 6. The compositionof claim 1, wherein the polyhydroxylated polymers are preformed naturalpolymers or hydroxyl-containing polymers selected from the groupconsisting of, multiply-hydroxylated polymers, polysaccharides,carbohydrates, polyols, polyvinyl alcohol, poly amino acids such aspolyserine, and other polymers such as 2-(hydroxyethyl)methacrylate. 7.The composition of claim 6, wherein the polyhydroxylated polymers arepolysaccharides.
 8. The composition of claim 7, wherein thepolysaccharides are selected from the group consisting of dextran,mannan, pullulan, maltodextrin, starches, cellulose and cellulosederivatives, gums, xanthan gum, locust bean gum, and pectin.
 9. Thecomposition of claim 8, wherein the polysaccharides are dextran ormannan.
 10. The composition of claim 1, wherein the modifiedpolyhydroxylated polymers are modified polysaccharides with pendantacetals, thus providing acetal-derivatized polysaccharides.
 11. Thecomposition of claim 10, wherein the acetal-derivatized polysaccharidesare acetal-derivatized dextran, acetal-derivatized mannan oracetal-derivatized polyvinyl alcohols.
 12. The composition of claim 1,wherein at least 20% to 85%, of the hydroxyl groups in the polymer aremodified.
 13. The composition of claim 2, wherein the functional grouprenders the modified polyhydroxylated polymer acid-degradable, pHsensitive and insoluble in water and soluble in common organic solventssuch as dichloromethane, tetrahydrofuran, or acetone.
 14. Thecomposition of claim 5, wherein the processed modified polyhydroxylatedpolymers further comprise a bioactive material conjugated or entrappedtherein.
 15. The composition of claim 14, wherein the bioactive materialis selected from the group consisting of polynucleotides, polypeptides,proteins, peptides, antibodies, vaccines, antigens, genetic agents,small molecule drugs, and therapeutic agents.
 16. The composition ofclaim 15, wherein the bioactive material is a polynucleotide.
 17. Thecomposition of claim 15, wherein the bioactive material is a therapeuticagent.
 18. The composition of claim 15, wherein the bioactive materialis a vaccine.
 19. An acid-degradable modified polyhydroxylated polymercomprising a polyhydroxylated polymer having reversibly-modified pendanthydroxyl groups, wherein the hydroxyl groups are modified to featureacid degradable, pH-sensitive functional groups, whereby the functionalgroups are designed to remain relatively stable in plasma at neutralphysiological pH (about 7.4), but degrade by hydrolysis in the moreacidic environment of about pH 5.0-6.0, thereby resulting in degradationupon hydrolysis.
 20. The acid-degradable modified polyhydroxylatedpolymer of claim 19 wherein the polyhydroxylated polymer is apolysaccharide.
 21. The composition of claim 20, wherein thepolysaccharides are dextran or mannan.
 22. The acid-degradable modifiedpolyhydroxylated polymer of claim 20, wherein the polymer is processedto deliver a bioactive material, whereupon the bioactive material isreleased in response to mildly acidic conditions, found in the body suchas in tumors, inflammatory tissues and in cellular compartments such aslysosomes and phagolysosomes of antigen presenting cells.
 23. Theacid-degradable modified polyhydroxylated polymer of claim 22, whereinthe bioactive material is selected from the group comprising ofantigens, proteins, polynucleotides, polypeptides, small drug moleculesand other bioactive material having a physiological effect on a cell.24. The acid-degradable modified polyhydroxylated polymer of claim 22,wherein the polymer is processed to form particles for delivery to acell.
 25. The acid-degradable modified polyhydroxylated polymer of claim24, wherein the particle size is 10 nm to 50 μm.
 26. The acid-degradablemodified polyhydroxylated polymer of claim 25, wherein the particle sizeis 10 nm to 2000 nm.
 27. The acid-degradable modified polyhydroxylatedpolymer of claim 26, wherein the particle size is 40 to 300 nm.
 28. Theacid-degradable modified polyhydroxylated polymer of claim 24, whereinthe polymer is processed to form or coat sutures, scaffolds, stents andother implantable polymer materials.
 29. The acid-degradable modifiedpolyhydroxylated polymer of claim 22, wherein the bioactive materialsare encapsulated in or conjugated to the polymer in bulk materials,scaffolds, coated stents and other implantable devices.
 30. A method ofdelivering a bioactive material to a cellular interior, comprising:providing to the cell acid-degradable particles having a bioactivematerial bound within or conjugated to an acid-degradable modifiedpolyhydroxylated polymer, whereby hydrolysis within an acidic cellularcompartment cleaves the acid-degradable linkage in said modifiedpolyhydroxylated polymer and releases said bioactive material.
 31. Thecomposition of claim 30 wherein the polyhydroxylated polymer is apolysaccharide.
 32. The composition of claim 31, wherein thepolysaccharide is dextran.
 33. The composition of claim 32, wherein thereagent used to modify dextran is a vinyl ether.
 34. The composition ofclaim 33 wherein the reagent used to modify dextran is 2-ethoxypropeneor 2-methoxypropene.
 35. The composition of claim 34, wherein thereagent used to modify dextran is 2-methoxypropene.
 36. The compositionof claim 30, wherein the bioactive material is selected from the groupconsisting of polynucleotides, polypeptides, proteins, peptides,antibodies, vaccines, antigens, genetic agents, small drugs, andtherapeutic agents.
 37. A method of preparing a modifiedpolyhydroxylated acid-degradable composition for delivering a bioactivematerial to a cell, comprising the steps of (a) preparing a mixturewhich contains a polyhydroxylated polymer and an acetal functionalgroup, wherein a one-step reaction provides an acetalatedpolyhydroxylated polymer having modified hydroxyl groups containing anacetal acid-degradable linkage; (b) forming particles of the polymer inthe presence of a bioactive material; and (c) recovering the resultingpolymer particles having bioactive material bound or entrapped thereto.38. The composition of claim 37 wherein the polyhydroxylated polymer isdextran.
 39. A composition comprising a modified polyhydroxylatedpolymer comprising a polyhydroxylated polymer having reversibly modifiedhydroxyl groups, whereby the hydroxyl groups are modified by anacid-catalyzed reaction between a polyhydroxylated polymer and a reagentselected from the group consisting of: acetals, aldehydes, vinyl ethersand ketones.
 40. The composition of claim 39 wherein thepolyhydroxylated polymer is modified under various reaction conditionsto yield a modified polymer with various degradation rates.
 41. Thecomposition of claim 40 wherein the polyhydroxylated polymer is apolysaccharide.
 42. The composition of claim 41 wherein thepolysaccharide is selected from the group consisting of dextran, mannan,pullulan, maltodextrin, starches, cellulose and cellulose derivatives,gums, xanthan gum, locust bean gum, and pectin.
 43. The composition ofclaim 42, wherein the polysaccharide is dextran.
 44. The composition ofclaim 39, wherein the reagent is a vinyl ether.
 45. The composition ofclaim 44 wherein the reagent is 2-ethoxypropene or 2-methoxypropene. 46.The composition of claim 45, wherein the reagent is 2-methoxypropene.47. The composition of claim 39, wherein said polymers are processed toform particles, bulk materials, tissue engineering scaffolds, orimplants.
 48. The acid-degradable modified polyhydroxylated polymer ofclaim 43, wherein the polymer is processed to deliver a bioactivematerial, whereupon the bioactive material is released in response tomildly acidic conditions, found in the body such as in tumors,inflammatory tissues and in cellular compartments such as lysosomes andphagolysosomes of antigen presenting cells.
 49. A composition comprisingan acetal modified polyhydroxylated processed polymer comprising apolyhydroxylated polymer having about 75-85% of its hydroxyl groupsreversibly modified to acetals, whereby said modification allows saidcomposition to be soluble in organic solvents and insoluble in water,wherein said modified polyhydroxylated polymer is processed intoparticles in the presence of a bioactive material.